Stable Solid Formulation of a GC-C Receptor Agonist Polypeptide Suitable for Oral Administration

ABSTRACT

Solid, stable formulations of linaclotide suitable for oral administration are described herein as are methods for preparing such formulations. The formulations described herein contain a polypeptide consisting of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (“linaclotide”; SEQ ID NO:1) or a pharmaceutically acceptable salt thereof. The linaclotide formulations described herein are stable and have a sufficient shelf life for manufacturing, storing and distributing the drug.

PRIORITY CLAIM

This application is a continuation of, and claims priority under 35U.S.C. §120 to U.S. patent application Ser. No. 12/541,410 filed Aug.14, 2009. This application also claims priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application No. 61/231,725 filed Aug. 6,2003; U.S. Provisional Patent Application No. 61/273,332 filed Aug. 3,2009; and U.S. Provisional Patent Application No. 61/089,422 filed Aug.15, 2008; entitled “Stable Solid Formulation of a GC-C Receptor AgonistPolypeptide Suitable for Oral Administration”. The entire contents ofthe aforementioned applications are incorporated herein by reference.

FIELD

This disclosure concerns solid formulations of a guanylate cyclase-Creceptor agonist polypeptide suitable for oral administration andmethods for preparing such formulations.

SEQUENCE LISTING

This application incorporates by reference in its entirety the SequenceListing entitled “IW057US1CON2_ST25.txt” (1.94 kilobytes), which wasrevised on May 21, 2014 and filed electronically herewith.

BACKGROUND

Many therapeutic polypeptides are formulated in aqueous solution becausethey are most active in this form. However, most polypeptides are notparticularly stable in aqueous solution, such that the formulationsoften have a short half-life and require refrigeration. Although aqueoussolutions of polypeptides can be dried by freeze-drying, spray-drying orother methods, such dried formulations may also be unstable and havereduced activity relative to an aqueous solution of the polypeptide.Typical break-down mechanisms that occur both in aqueous solution and indried formulations include aggregation and oxidative or hydrolyticdegradation. Thus, the majority of therapeutic polypeptides, whether inaqueous solution or dried, are stored under refrigerated conditions dueto their limited stability.

Linaclotide is a peptide having the amino acid sequence Cys Cys Glu TyrCys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 1) that activatesthe guanylate cyclase-C (GC-C) receptor. Linaclotide, which may beadministered orally, is useful for the treatment of gastrointestinaldisorders and conditions, including irritable bowel syndrome (IBS) andchronic constipation (CC). Formulations comprising linaclotide haveneeded to be refrigerated in order to avoid degradation over time.However, refrigeration is inconvenient both for commercial distributionof the drug and for storage by patients. Thus, there is a need to have asolid linaclotide formulation that is stable at room temperature for atleast 12 months.

SUMMARY

Solid, stable formulations of linaclotide suitable for oraladministration are described herein as are methods for preparing suchformulations. The formulations described herein contain a polypeptideconsisting of the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn ProAla Cys Thr Gly Cys Tyr (“linaclotide”; SEQ ID NO: 1) or apharmaceutically acceptable salt thereof.

The linaclotide formulations described herein are stable and have asufficient shelf life for manufacturing, storing and distributing thedrug. For example, formulations described herein are expected to have ashelf life of at least 12 months at room temperature storage conditions(e.g., 25° C./60% relative humidity (RH)). In further embodiments, theformulations described herein are expected to have a shelf life of atleast 18 months or at least 24 months at room temperature storageconditions (e.g., 25° C./60% RH).

In some embodiments, formulations are described wherein ≧95% of theoriginal amount of linaclotide in the composition remains after threemonths when packaged samples are stored at accelerated conditions (40°C./75% RH) when assessed in an assay on a weight/weight basis asdetermined by high pressure liquid chromatography (HPLC) against alinaclotide reference standard. In further embodiments, ≧90% of theoriginal amount of linaclotide in the composition remains after at least6 months when packaged samples are stored at accelerated conditions (40°C./75% RH). In other embodiments, formulations are described whereinchromatographic purity of the linaclotide as determined as area percentby HPLC remains at ≧95% over the course of at least three months whenpackaged samples are stored at accelerated conditions (40° C./75% RH).In further embodiments, the chromatographic purity of the linaclotide asdetermined by area percent by HPLC remains at ≧90% over the course of atleast 6 months when packaged samples are stored at acceleratedconditions (40° C./75% RH). Thus, for example, no more than about 10% ofthe linaclotide undergoes degradation to other products such as anoxidation product of linaclotide, a hydrolysis product of linaclotide ora formaldehyde-mediated imine product of linaclotide (“formaldehydeimine product”).

In one embodiment, the invention comprises a pharmaceutical compositioncomprising linaclotide, wherein the chromatographic purity of thelinaclotide decreases by less than 10% after 18 months or 24 months ofstorage of the pharmaceutical composition at 25° C. at 60% relativehumidity in a sealed container containing a desiccant. In a furtherembodiment, the chromatographic purity of the linaclotide decreases byless than 9%, 8%, 7%, 6%, 5%, 4% or 2% after 18 months or 24 months ofstorage of the pharmaceutical composition at 25° C. at 60% relativehumidity in a sealed container containing a desiccant. In anotherembodiment, the invention comprises a pharmaceutical compositioncomprising linaclotide, wherein the chromatographic purity of thelinaclotide decreases by less than 10% after 3 months or 6 months ofstorage of the pharmaceutical composition at 40° C. at 75% relativehumidity in a sealed container containing a desiccant. In a furtherembodiment, the chromatographic purity of the linaclotide decreases byless than 9%, 8%, 7%, 6%, 5%, 4% or 2% after 3 months or 6 months ofstorage of the pharmaceutical composition at 40° C. at 75% relativehumidity in a sealed container containing a desiccant.

In one embodiment, the invention comprises a unit dosage form of apharmaceutical composition comprising linaclotide, wherein thechromatographic purity of the linaclotide decreases by less than 10%after 18 months or 24 months of storage of the unit dosage form at 25°C. at 60% relative humidity in a sealed container containing adesiccant. In a further embodiment, the chromatographic purity of thelinaclotide decreases by less than 9%, 8%, 7%, 6%, 5%, 4% or 2% after 18months or 24 months of storage of the unit dosage form at 25° C. at 60%relative humidity in a sealed container containing a desiccant. Inanother embodiment, the invention comprises a unit dosage form of apharmaceutical composition comprising linaclotide, wherein thechromatographic purity of the linaclotide decreases by less than 10%after 3 months or 6 months of storage of the unit dosage form at 40° C.at 75% relative humidity in a sealed container containing a desiccant.In a further embodiment, the chromatographic purity of the linaclotidedecreases by less than 9%, 8%, 7%, 6%, 5%, 4% or 2% after 3 months or 6months of storage of the unit dosage form at 40° C. at 75% relativehumidity in a sealed container containing a desiccant.

In one embodiment, the invention comprises a sealed container comprisinga plurality of unit dosage forms of a pharmaceutical compositioncomprising linaclotide, wherein the chromatographic purity of thelinaclotide decreases by less than 10% after 18 months or 24 months ofstorage of the sealed container containing a desiccant at 25° C. at 60%relative humidity. In a further embodiment, the chromatographic purityof the linaclotide decreases by less than 9%, 8%, 7%, 6%, 5%, 4% or 2%after 18 months or 24 months of storage of the sealed containercontaining a desiccant at 25° C. at 60% relative humidity. In anotherembodiment, the invention comprises a sealed container comprising aplurality of unit dosage forms of a pharmaceutical compositioncomprising linaclotide, wherein the chromatographic purity of thelinaclotide decreases by less than 10% after 3 months or 6 months ofstorage of the sealed container containing a desiccant at 40° C. at 75%relative humidity. In a further embodiment, the chromatographic purityof the linaclotide decreases by less than 9%, 8%, 7%, 6%, 5%, 4% or 2%after 3 months or 6 months of storage of the sealed container containinga desiccant at 40° C. at 75% relative humidity.

In one embodiment, the invention comprises a pharmaceutical compositioncomprising linaclotide, wherein the assay value for linaclotidedetermined on a weight/weight basis decreases by less than 10% after 18months or 24 months of storage of the pharmaceutical composition at 25°C. at 60% relative humidity in a sealed container containing adesiccant. In a further embodiment, the assay value for linaclotidedetermined on a weight/weight basis decreases by less than 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% after 18 months or 24 months of storage of thepharmaceutical composition at 25° C. at 60% relative humidity in asealed container containing a desiccant. In another embodiment, theinvention comprises a pharmaceutical composition comprising linaclotide,wherein the assay value for linaclotide determined on a weight/weightbasis decreases by less than 10% after 3 months or 6 months of storageof the pharmaceutical composition at 40° C. at 75% relative humidity ina sealed container containing a desiccant. In a further embodiment, thechromatographic purity of the linaclotide decreases by less than 9%, 8%,7%, 6%, 5%, 4%, 3%, 2% or 1% after 3 months or 6 months of storage ofthe pharmaceutical composition at 40° C. at 75% relative humidity in asealed container containing a desiccant.

In one embodiment, the invention comprises a unit dosage form of apharmaceutical composition comprising linaclotide, wherein the assayvalue for linaclotide determined on a weight/weight basis decreases byless than 10% after 18 months or 24 months of storage of the unit dosageform at 25° C. at 60% relative humidity in a sealed container containinga desiccant. In a further embodiment, the assay value for linaclotidedetermined on a weight/weight basis decreases by less than 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% after 18 months or 24 months of storage of theunit dosage form at 25° C. at 60% relative humidity in a sealedcontainer containing a desiccant. In another embodiment, the inventioncomprises a unit dosage form of a pharmaceutical composition comprisinglinaclotide, wherein the assay value for linaclotide determined on aweight/weight basis decreases by less than 10% after 3 months or 6months of storage of the unit dosage form at 40° C. at 75% relativehumidity in a sealed container containing a desiccant. In a furtherembodiment, the assay value for linaclotide determined on aweight/weight basis decreases by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%,2% or 1% after 3 months or 6 months of storage of the unit dosage format 40° C. at 75% relative humidity in a sealed container containing adesiccant.

In one embodiment, the invention comprises a sealed container comprisinga plurality of unit dosage forms of a pharmaceutical compositioncomprising linaclotide, wherein the assay value for linaclotidedetermined on a weight/weight basis decreases by less than 10% after 18months or 24 months of storage of the sealed container at 25° C. at 60%relative humidity in a sealed container containing a desiccant. In afurther embodiment, the assay value for linaclotide determined on aweight/weight basis decreases by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%,2% or 1% after 18 months or 24 months of storage of the sealed containercontaining a desiccant at 25° C. at 60% relative humidity. In anotherembodiment, the invention comprises a sealed container comprising aplurality of unit dosage forms of a pharmaceutical compositioncomprising linaclotide, wherein the assay value for linaclotidedetermined on a weight/weight basis decreases by less than 10% after 3months or 6 months of storage of the sealed container containing adesiccant at 40° C. at 75% relative humidity. In a further embodiment,the assay value for linaclotide determined on a weight/weight basisdecreases by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% after 3months or 6 months of storage of the sealed container containing adesiccant at 40° C. at 75% relative humidity.

In some embodiments, there is provided a pharmaceutical compositioncomprising linaclotide and a hydrolysis product comprising:

In some embodiments, the hydrolysis product comprises less than about15% by weight of the composition, less than about 10% by weight of thecomposition, less than about 7% by weight of the composition or lessthan about 5% by weight of the composition. In other embodiments, thehydrolysis product comprises from about 0.01% to about 15% by weight ofthe composition, about 0.05% to about 10% by weight of the composition,about 0.05% to about 7% by weight of the composition or about 0.05% toabout 5% by weight of the composition. In further embodiments, there isprovided a method of treating a gastrointestinal disorder in a patientin need thereof comprising administering a pharmaceutical compositioncomprising linaclotide and a hydrolysis product.

In some embodiments, there is provided a pharmaceutical compositioncomprising linaclotide and a formaldehyde imine product comprising:

In some embodiments, the formaldehyde imine product comprises less thanabout 15% by weight of the composition, less than about 10% by weight ofthe composition, less than about 7% by weight of the composition or lessthan about 5% by weight of the composition. In other exemplaryembodiments, the formaldehyde imine product comprises from about 0.01%to about 15% by weight of the composition, about 0.05% to about 10% byweight of the composition, about 0.05% to about 7% by weight of thecomposition or about 0.05% to about 5% by weight of the composition. Infurther embodiments, there is provided a method of treating agastrointestinal disorder in a patient in need thereof comprisingadministering a pharmaceutical composition comprising linaclotide and aformaldehyde imine product.

In some embodiments, there is provided a pharmaceutical compositioncomprising linaclotide and a linaclotide oxidation product. In oneembodiment, the linaclotide oxidation product has a molecular weight of1542.8, which most likely forms as the addition of a single oxygen atomto one of the six cysteinyl sulfurs in linaclotide. One potentialstructure of the product is depicted below, although one of skill in theart will recognize that the oxygen atom may be attached to any of theother five sulfurs:

In another embodiment, there may be an addition of more than one oxygenatom to linaclotide, which would increase its molecular weight by 16 AUper added oxygen atom.

In some embodiments, the linaclotide oxidation product comprises lessthan about 15% by weight of the composition, less than about 10% byweight of the composition, less than about 7% by weight of thecomposition or less than about 5% by weight of the composition. In otherexemplary embodiments, the linaclotide oxidation product comprises fromabout 0.01% to about 15% by weight of the composition, about 0.05% toabout 10% by weight of the composition, about 0.05% to about 7% byweight of the composition or about 0.05% to about 5% by weight of thecomposition. In further embodiments, there is provided a method oftreating a gastrointestinal disorder in a patient in need thereofcomprising administering a pharmaceutical composition comprisinglinaclotide and a linaclotide oxidation product.

The assay value on a weight/weight basis (“weight/weight assay”) may bedetermined by comparing, e.g., by HPLC, the amount of linaclotide in asample, to a linaclotide reference standard. As used herein, the weightof linaclotide in a composition after storage at room temperature oraccelerated conditions at a specified time point (e.g., three or sixmonths of storage under accelerated conditions [40° C./75% RH] or 12, 18or 24 months of storage under room temperature conditions [25° C./60%RH]) is compared to the weight of linaclotide in a composition at aninitial time (e.g., the time when the pharmaceutical composition isreleased for clinical or patient use (“the release date”)) to providethe weight/weight assay value. For example, the weight of linaclotide ina composition is measured after storage for a specified time ataccelerated conditions (40° C./75% RH) and compared to the weight oflinaclotide that was present in the sample at the release date. Inanother example, the weight of linaclotide in a composition is measuredafter storage for a specified time at room temperature conditions (25°C./60% RH) and compared to the weight of linaclotide that was present inthe sample at the release date. Thus, the phrase “≧90% of the originalamount of linaclotide in the composition remains after at least 6 monthswhen packaged samples are stored at accelerated conditions (40° C./75%RH)” means the weight of linaclotide in the composition measured in anassay on a weight/weight basis as determined by HPLC after at least 6months storage at accelerated conditions is ≧90% of the amount oflinaclotide in the composition present at the initial time (e.g., therelease date of the linaclotide composition).

Chromatographic purity of linaclotide may be assessed by performing HPLCunder the conditions described herein. The area under the linaclotidepeak is measured and compared to the total area under all peaksexcluding the solvent peak and any non-polypeptide related peaks (i.e.,peaks associated with excipients that may be observed in a placebo). Asused herein, the chromatographic purity of linaclotide in a compositionafter storage at room temperature or accelerated conditions at aspecified time point (e.g., three or six months of storage underaccelerated conditions [40° C./75% RH] or 12, 18 or 24 months of storageunder room temperature conditions [25° C./60% RH]) is compared to thechromatographic purity of linaclotide in a composition at an initialtime (e.g., the time when the pharmaceutical composition is released forclinical or patient use (“the release date”)) to provide thechromatographic purity value. For example, the chromatographic purity oflinaclotide in a composition is measured after storage for a specifiedtime at accelerated conditions (40° C./75% RH) and compared to thechromatographic purity of linaclotide in the composition at the releasedate. In another example, the chromatographic purity of linaclotide in acomposition is measured after storage for a specified time at roomtemperature conditions (25° C./60% RH) and compared to thechromatographic purity of linaclotide in the composition at the releasedate.

This disclosure features a method for preparing a pharmaceuticalcomposition comprising linaclotide or a pharmaceutically acceptable saltthereof, the method comprising: (a) providing a solution, e.g., anaqueous solution (“the coating solution”), comprising: (i) linaclotideor a pharmaceutically acceptable salt thereof; (ii) a cation selectedfrom Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na or Al³⁺ and/or a sterically hinderedprimary amine (e.g., leucine) and, optionally, (iii) a pharmaceuticallyacceptable binder; and (b) applying the coating solution to apharmaceutically acceptable filler to generate polypeptide-coated filler(e.g., by spraying, mixing or coating the pharmaceutically acceptablefiller with the coating solution). The method can optionally include oneor more of: (i) blending the polypeptide-coated filler with apharmaceutically acceptable glidant, a pharmaceutically acceptablelubricant or a pharmaceutically acceptable additive that acts as both aglidant and lubricant; (ii) blending the polypeptide-coated filler withfiller that is not polypeptide-coated, (iii) blending thepolypeptide-coated filler with other additives; (iii) applying apharmaceutically acceptable coating additive to the polypeptide-coatedfiller. The final pharmaceutical composition can be placed into capsules(e.g., gelatin capsule) or used to form tablets.

It has been found that a cation selected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺,K⁺, Na⁺ or Al³⁺ is useful for suppressing the formation of an oxidationproduct of linaclotide during storage. It has also been found that asterically hindered primary amine, e.g., leucine, is useful forsuppressing the formation of a formaldehyde imine adduct of linaclotide(“formaldehyde imine product”) during storage. Thus, a linaclotideformulation comprising a cation selected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺,K⁺, Na⁺ or Al³⁺ (e.g., a divalent cation selected from Zn²⁺, Mg²⁺ orCa²⁺) and/or a sterically hindered primary amine, such as an amino acid,has a sufficient shelf life (as measured by chromatographic purityand/or by a weight/weight assay) for manufacturing, storing anddistributing the drug. Further, while the presence of a stericallyhindered amine alone can increase the formation of a hydrolysis productof linaclotide during storage, the combination of a sterically hinderedprimary amine and a cation, e.g., the combination of leucine and Ca²⁺,suppresses the formation of the hydrolysis product of linaclotide aswell as the oxidation product of linaclotide during storage, leading toan even greater overall stability as determined by a weight/weight assayand/or by chromatographic purity.

In some embodiments, there is provided a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, linaclotide and one ormore agents selected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na or Al³⁺ and asterically hindered primary amine, wherein the agent improves at leastone attribute of the composition, relative to a pharmaceuticalcomposition without said agent. In further embodiments, the agent isMg²⁺, Ca²⁺ or Zn²⁺. In a further embodiment, the agent is Ca²⁺. In someembodiments, the cation is provided as, without limitation, magnesiumacetate, magnesium chloride, magnesium phosphate, magnesium sulfate,calcium acetate, calcium chloride, calcium phosphate, calcium sulfate,zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganeseacetate, manganese chloride, manganese phosphate, manganese sulfate,potassium acetate, potassium chloride, potassium phosphate, potassiumsulfate, sodium acetate, sodium chloride, sodium phosphate, sodiumsulfate, aluminum acetate, aluminum chloride, aluminum phosphate oraluminum sulfate. In further embodiments, the cation is provided asmagnesium chloride, calcium chloride, calcium phosphate, calciumsulfate, zinc acetate, manganese chloride, potassium chloride, sodiumchloride or aluminum chloride. In other embodiments, the cation isprovided as calcium chloride, magnesium chloride or zinc acetate.

In another embodiment, the agent is a sterically hindered primary amine.In a further embodiment, the sterically hindered primary amine is anamino acid. In yet a further embodiment, the amino acid is anaturally-occurring amino acid. In a still further embodiment, thenaturally-occurring amino acid is selected from the group consisting ofhistidine, phenylalanine, alanine, glutamic acid, aspartic acid,glutamine, leucine, methionine, asparagine, tyrosine, threonine,isoleucine, tryptophan, methionine and valine; yet further, thenaturally-occurring amino acid is leucine, isoleucine, alanine ormethionine; in another embodiment, the naturally-occurring amino acid isleucine or methionine; still further, the naturally-occurring amino acidis leucine. In another embodiment, the sterically hindered primary amineis a non-naturally occurring amino acid or amino acid derivative (e.g.,1-aminocyclohexane carboxylic acid, lanthionine or theanine). In afurther embodiment, the sterically hindered primary amine iscyclohexylamine, 2-methylbutylamine or chitosan.

In other embodiments, there is provided a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, linaclotide, a cationselected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ (e.g., a divalentcation selected from Zn²⁺, Mg²⁺ or Ca²⁺) and a sterically hinderedprimary amine. In one embodiment, the cation is Ca²⁺. In anotherembodiment, the cation is a mixture of two or three of Mg²⁺, Ca²⁺, Zn²⁺,Mn²⁺, K⁺, Na⁺ or Al³⁺ (e.g., a mixture of two or three of Zn²⁺, Mg²⁺ orCa². In a further embodiment, the pharmaceutical composition furthercomprises a pharmaceutically acceptable binder and/or a pharmaceuticallyacceptable glidant, lubricant or additive that acts as both a glidantand lubricant and/or an antioxidant. In a further embodiment, thesterically hindered primary amine is an amino acid. In yet a furtherembodiment, the amino acid is a naturally-occurring amino acid. In astill further embodiment, the naturally-occurring amino acid is selectedfrom the group consisting of: histidine, phenylalanine, alanine,glutamic acid, aspartic acid, glutamine, leucine, methionine,asparagine, tyrosine, threonine, isoleucine, tryptophan, methionine andvaline; yet further, the naturally-occurring amino acid is leucine,isoleucine, alanine or methionine; in another embodiment, thenaturally-occurring amino acid is leucine or methionine; still further,the naturally-occurring amino acid is leucine. In another embodiment,the sterically hindered primary amine can be a mixture of more than onesterically hindered primary amines. For example, the sterically hinderedprimary amine may be a mixture of two or more sterically hinderedprimary amines, e.g., a mixture of two or more amino acids.

In some cases the molar ratio of cation: sterically hindered primaryamine:linaclotide (e.g., Ca²⁺:leucine:linaclotide) in the aqueoussolution applied to the carrier is 5-100:5-50:1. It can be desirable forthe molar ratio of cation:sterically hindered primary amine (e.g.,Ca²⁺:leucine) to be equal to or greater than 2:1 (e.g., between 5:1 and2:1). Thus, in some cases the molar ratio of cation: sterically hinderedprimary amine:linaclotide (e.g., Ca²⁺:leucine:linaclotide) applied tothe carrier is 100:50:1, 100:30:1, 80:40:1, 80:30:1, 80:20:1, 60:30:1,60:20:1, 50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1, 10:5:1 or 5:10:1.When binder, e.g., methylcellulose, is present in the linaclotidesolution applied to the carrier it can be present at 0.5%-2.5% by weight(e.g., 0.7%-1.7% or 0.7%-1% or 1.5% or 0.7%).

The weight of linaclotide applied to a given weight of filler (e.g.,microcrystalline cellulose) can vary from about 0.02:100 to about2.67:100. Thus, about 0.05 mg to about 6.0 mg of linaclotide can beapplied to 225 mg of filler. In a further embodiment, the weight oflinaclotide applied to a given weight of filler is about 0.05 mg toabout 2.0 mg of linaclotide (e.g., 0.1, 0.2, 0.3. 0.4, 0.5, 0.6, 0.7 mgpeptide for 225 mg of filler).

In various embodiments: the sterically hindered primary amine is anamino acid (e.g., a naturally-occurring amino acid or anaturally-occurring amino acid selected from histidine, phenylalanine,alanine, glutamic acid, aspartic acid, glutamine, methionine,asparagine, tyrosine, threonine, leucine, isoleucine, tryptophan, orvaline). In other cases the sterically hindered primary amine is anon-naturally occurring amino acid or amino acid derivative (e.g.,lanthionine, theanine or 1-amino cyclohexane). In other cases, thesterically hindered primary amine is an amino sugar (e.g., chitosan orglucosamine).

In some cases, the sterically hindered primary amine has the formula:

wherein R₁, R₂ and R₃ are independently selected from: H; —C(O)OH; C1-C6alkyl, optionally substituted by —CO₂H, —CONH₂, or a 5-10 membered arylor heteroaryl; C1-C6 alkoxyalkyl; or C1-C6 thioalkoxyalkyl, wherein anyof the alkyl or aryl groups above can be singly or multiply substitutedwith halogen or —NH₂, and provided that no more than two of R₁, R₂ andR₃ are H. In a further embodiment, no more than one of R₁, R₂ and R₃ isH.

The term “alkyl”, as used herein, refers to a saturated linear orbranched-chain monovalent hydrocarbon radical. Unless otherwisespecified, an alkyl group contains 1-20 carbon atoms (e.g., 1-20 carbonatoms, 1-10 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, 1-4 carbonatoms or 1-3 carbon atoms). Examples of alkyl groups include, but arenot limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,s-butyl, t-butyl, pentyl, hexyl, heptyl, octyl and the like.

The terms C_(n-m)“alkoxyalkyl” and C_(n-m) “thioalkoxyalkyl” mean alkyl,substituted with one or more alkoxy or thioalkoxy groups, as the casemay be, wherein the combined total number of carbons of the alkyl andalkoxy groups, or alkyl and thioalkoxy groups, combined, as the case maybe, is between the values of n and m. For example, a C₄₋₆ alkoxyalkylhas a total of 4-6 carbons divided between the alkyl and alkoxy portion;e.g. it can be —CH₂OCH₂CH₂CH₃, —CH₂CH₂OCH₂CH₃ or —CH₂CH₂CH₂OCH₃.

As used herein, the term “aryl” (as in “aryl ring” or “aryl group”),used alone or as part of a larger moiety, refers to a carbocyclic ringsystem wherein at least one ring in the system is aromatic and has asingle point of attachment to the rest of the molecule. Unless otherwisespecified, an aryl group may be monocyclic, bicyclic or tricyclic andcontain 6-18 ring members. Examples of aryl rings include, but are notlimited to, phenyl, naphthyl, indanyl, indenyl, tetralin, fluorenyl, andanthracenyl.

The term “heteroaryl” (or “heteroaromatic” or “heteroaryl group” or“aromatic heterocycle”) used alone or as part of a larger moiety as in“heteroarallyl” or “heteroarylalkoxy” refers to a ring system wherein atleast one ring in the system is aromatic and contains one or moreheteroatoms, wherein each ring in the system contains 3 to 7 ringmembers and which has a single point of attachment to the rest of themolecule. Unless otherwise specified, a heteroaryl ring system may bemonocyclic, bicyclic or tricyclic and have a total of five to fourteenring members. In one embodiment, all rings in a heteroaryl system arearomatic. Also included in this definition are heteroaryl radicals wherethe heteroaryl ring is fused with one or more aromatic or non-aromaticcarbocyclic or heterocyclic rings, or combinations thereof, as long asthe radical or point of attachment is in the heteroaryl ring. Bicyclic6,5 heteroaromatic system, as used herein, for example, is a sixmembered heteroaromatic ring fused to a second five membered ringwherein the radical or point of attachment is on the six membered ring.

Heteroaryl rings include, but are not limited to the followingmonocycles: 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl,4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl(e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl(e.g., 2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyrazinyl, 1,3,5-triazinyl, andthe following bicycles: benzimidazolyl, benzofuryl, benzothiophenyl,benzopyrazinyl, benzopyranonyl, indolyl (e.g., 2-indolyl), purinyl,quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl), andisoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl, or4-isoquinolinyl).

In various cases: the antioxidant is selected from BHA (butylatedhydroxyanisole), BHT (butylated hydroxytoluene), vitamin E, propylgallate, ascorbic acid and salts or esters thereof, tocopherol andesters thereof, alpha-lipoic acid, beta-carotene; the pharmaceuticallyacceptable binder is polyvinyl alcohol or polyvinyl pyrrolidone; thepharmaceutically acceptable binder is selected from: a starch (e.g.,corn starch, pre-gelatinized potato starch, rice starch, wheat starch,and sodium starch glycollate), maltodextrin or a cellulose ether (e.g.,methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxyethyl methylcellulose, hydroxypropyl cellulose andhydroxypropyl methylcellulose); the pharmaceutically acceptable filleris cellulose (e.g., microfine cellulose or microcrystalline cellulosesuch as Celphere CP-305 or Avicel); the pharmaceutically acceptablefiller is a sugar or a sugar alcohol (e.g., mannitol, isomalt, sorbitol,dextrose, xylitol, sucrose and lactose); the filler comprises particleshaving an average diameter between 50 μm and 1000 μm; the lubricantand/or glidant is selected from: talc, leucine, magnesium stearate,stearic acid and polyvinyl alcohol; and the lubricant and/or glidant isselected from: calcium stearate, mineral oil, vegetable oil,polyethylene glycol (PEG; e.g., PEG that is liquid or solid at roomtemperature), sodium benzoate, and sodium lauryl sulfate.

In some cases, the linaclotide solution used in a method for preparingthe formulation has a pH below 7 (e.g., a pH between 1 and 3 or a pHbetween about 1.5 and about 2.5). The pH can be adjusted with, e.g.,phosphoric acid. In some cases, the solution is buffered. Variouspharmaceutically acceptable buffers can be used (e.g., phosphatebuffer).

In some cases, the linaclotide solution used in a method for preparingthe formulation comprises both a cation (e.g., CaCl₂) and a stericallyhindered primary amine (e.g., leucine).

In some cases the linaclotide solution comprises CaCl₂ and leucine; thebinder is methylcellulose; the filler is microcrystalline cellulose; theglidant and/or lubricant comprises talc or leucine.

Also provided is a pharmaceutical composition prepared by any of themethods described herein.

In another aspect, a pharmaceutical composition is disclosed thatcomprises a pharmaceutically acceptable carrier, linaclotide and one ormore agents selected from (i) a cation selected from Mg²⁺, Ca²⁺, Zn²⁺,Mn²⁺, K⁺, Na⁺ or Al³⁺, or (ii) a sterically hindered primary amine. Insome embodiments, the pharmaceutical composition comprises at least onecation and at least one sterically hindered primary amine.

Methods of using the pharmaceutical compositions to treat a variety ofgastrointestinal disorders are also described.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 demonstrates an example of an analysis of linaclotide by HPLC,wherein “Oxidation” refers to the linaclotide oxidation product,“Formaldehyde Imine” refers to the linaclotide formaldehyde imineproduct and “Hydrolysis” refers to the linaclotide hydrolysis product.

This FIGURE is provided by way of example and is not intended to limitthe scope of the present invention.

DETAILED DESCRIPTION

Oral compositions containing linaclotide can be used to treat a varietyof gastrointestinal disorders. In various embodiments, the patient issuffering from a gastrointestinal disorder; the patient is sufferingfrom a disorder selected from the group consisting of: gastrointestinalmotility disorders, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction, Crohn's disease, duodenogastric reflux, dyspepsia,functional dyspepsia, nonulcer dyspepsia, a functional gastrointestinaldisorder, functional heartburn, gastroesophageal reflux disease (GERD),gastroparesis, irritable bowel syndrome, post-operative ileus,ulcerative colitis, chronic constipation, constipation, pain associatedwith constipation, and disorders and conditions associated withconstipation (e.g. constipation associated with use of opiate painkillers, post-surgical constipation, and constipation associated withneuropathic disorders as well as other conditions and disordersdescribed herein); the patient is suffering from a gastrointestinalmotility disorder, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction, Crohn's disease, duodenogastric reflux, dyspepsia,functional dyspepsia, nonulcer dyspepsia, a functional gastrointestinaldisorder, functional heartburn, gastroesophageal reflux disease (GERD),gastroparesis, inflammatory bowel disease, irritable bowel syndrome(e.g. diarrhea-predominant irritable bowel syndrome (d-IBS),constipation-predominant irritable bowel syndrome (c-IBS) and/oralternating irritable bowel syndrome (a-IBS)), post-operative ileus,ulcerative colitis, chronic constipation, constipation, pain associatedwith constipation, and disorders and conditions associated withconstipation (e.g. constipation associated with use of opiate painkillers, post-surgical constipation, and constipation associated withneuropathic disorders as well as other conditions and disordersdescribed herein); the patient has been diagnosed with a functionalgastrointestinal disorder according to the Rome Criteria (e.g. Rome II),the patient has been diagnosed with irritable bowel syndrome (e.g. (e.g.diarrhea predominant-IBS, constipation predominant-IBS, and/oralternating-IBS), according to the Rome Criteria (e.g. Rome II).

The dose range of linaclotide for adult humans is generally from 25 μgto 6 mg per day orally. In a further embodiment, the dose range is 25 μgto 2 mg per day orally. In some embodiments, the dose range for adulthumans is 50 μg to 1 mg per day orally (e.g., 50 μg, 67.5 μg, 100 μg,133 μg, 150 μg, 200 μg, 250 μg, 266 μg, 300 μg, 350 μg, 400 μg, 450 μg,500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg,950 μg or 1 mg). In further embodiments, the dose range is 100 μg to 600μg per day orally. In other embodiments, the dose is 50 μg, 67.5 μg, 100μg, 133 μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg or 600 μglinaclotide per day orally. In one embodiment, the linaclotidecomposition is provided in a discrete unit, a unit dosage form, (e.g., atablet, a capsule, a sachet) that is effective at such dosage or as amultiple of the same. In certain embodiments, the unit dosage form anddaily dose are equivalent. In various embodiments, the unit dosage formis administered with food at anytime of the day, without food at anytimeof the day, with food after an overnight fast (e.g. with breakfast). Invarious embodiments, the unit dosage form is administered once a day,twice a day or three times a day. The unit dosage form can optionallycomprise other additives. In some embodiments, one, two or three unitdosage forms will contain the daily oral dose of linaclotide. Theprecise amount of compound administered to a patient will be theresponsibility of the attendant physician. However, the dose employedwill depend on a number of factors, including the age and sex of thepatient, the precise disorder being treated, and its severity.

In one embodiment, there is provided a method for treating irritablebowel syndrome with constipation (IBS-c) in an adult patient in needthereof, comprising administering to the patient once daily an effectiveamount of a pharmaceutical composition described herein. In variousembodiments, the pharmaceutical composition comprises 133 μg or 266 μglinaclotide per unit dose per day. In other embodiments, thepharmaceutical composition is administered for a period of at least oneday, two days, three days, four days, five days, six days, one week, twoweeks, three weeks, four weeks or longer. In some embodiments, treatmentwith the linaclotide composition improves at least one symptom selectedfrom reduced abdominal pain, an increase in the number of completespontaneous bowel movements (CSBM) in a week, an increase in the numberof spontaneous bowel movements (SBM) in a week, improved stoolconsistency, reduced straining, reduced abdominal discomfort, reducedbloating or reduced IBS-c symptom severity.

In one embodiment, there is provided a method for treating chronicconstipation in an adult patient in need thereof, comprisingadministering to the patient once daily an effective amount of apharmaceutical composition described herein. In various embodiments, thepharmaceutical composition comprises 133 μg or 266 μg linaclotide perunit dose per day. In other embodiments, the pharmaceutical compositionis administered for a period of at least one day, two days, three days,four days, five days, six days, one week, two weeks, three weeks, fourweeks or longer. In some embodiments, treatment with the linaclotidecomposition improves at least one symptom selected from an increase inthe number of complete spontaneous bowel movements (CSBM) in a week, anincrease in the number of spontaneous bowel movements (SBM) in a week,improved stool consistency, reduced straining, reduced abdominaldiscomfort, reduced bloating or reduced severity of constipation.

Stool consistency of each BM may be monitored by the 7-point BristolStool Form Scale (BSFS) (1=hard lumps, 2=lumpy sausage, 3=crackedsausage, 4=smooth sausage, 5=soft lumps, 6=mushy, 7=watery). Strainingmay be monitored by the 7-point Ease of Passage Scale (1=manualdisimpaction/enema needed, 2=severe straining, 3=moderate straining,4=mild straining, 5=no straining, 6=urgency, 7=incontinent). CSBM may bemeasured by the sensation of complete emptying after an SBM (yes/no).Abdominal discomfort, bloating and severity of constipation may bemeasured using, e.g., a 5-point ordinal scale (1=none, 2=mild,3=moderate, 4=severe, 5=very severe).

A cation of the invention may be provided as a pharmaceuticallyacceptable salt i.e., a cation with an appropriate counterion. Examplesof pharmaceutically acceptable salts that may be used in the inventioninclude, without limitation, magnesium acetate, magnesium chloride,magnesium phosphate, magnesium sulfate, calcium acetate, calciumchloride, calcium phosphate, calcium sulfate, zinc acetate, zincchloride, zinc phosphate, zinc sulfate, manganese acetate, manganesechloride, manganese phosphate, manganese sulfate, potassium acetate,potassium chloride, potassium phosphate, potassium sulfate, sodiumacetate, sodium chloride, sodium phosphate, sodium sulfate, aluminumacetate, aluminum chloride, aluminum phosphate or aluminum sulfate. Insome embodiments, the pharmaceutically acceptable salts include calciumchloride, calcium carbonate, calcium acetate, magnesium chloride,magnesium acetate, zinc acetate and zinc chloride. In furtherembodiments, a pharmaceutically acceptable salt that may be used iscalcium chloride, magnesium chloride and zinc acetate.

As used herein, the term “binder” refers to any pharmaceuticallyacceptable binder that may be used in the practice of the invention.Examples of pharmaceutically acceptable binders include, withoutlimitation, a starch (e.g., corn starch, potato starch andpre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM®, sold byColorcon, Ltd.) and other starches), maltodextrin, gelatin, natural andsynthetic gums such as acacia, powdered tragacanth, guar gum, celluloseand its derivatives (e.g., methylcellulose, hydroxyethyl cellulose,hydroxyethyl methylcellulose, hydroxypropyl cellulose and hydroxypropylmethylcellulose (hypromellose), ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose,carboxymethylcellulose, microcrystalline cellulose (e.g. AVICEL™, suchas, AVICEL-PH-101™, -103™ and -105™, sold by FMC Corporation, MarcusHook, Pa., USA)), polyvinyl alcohol, polyvinyl pyrrolidone (e.g.,polyvinyl pyrrolidone K30), and mixtures thereof.

As used herein, the term “filler” refers to any pharmaceuticallyacceptable filler that may be used in the practice of the invention.Examples of pharmaceutically acceptable fillers include, withoutlimitation, talc, calcium carbonate (e.g., granules or powder), dibasiccalcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g.,granules or powder), microcrystalline cellulose (e.g., AVICEL PH101 orCELPHERE CP-305), powdered cellulose, dextrates, kaolin, mannitol,silicic acid, sorbitol, starch (e.g., Starch 1500), pre-gelatinizedstarch, lactose, glucose, fructose, galactose, trehalose, sucrose,maltose, isomalt, raffinose, maltitol, melezitose, stachyose, lactitol,palatinite, xylitol, myoinositol, and mixtures thereof.

Examples of pharmaceutically acceptable fillers that may be particularlyused for coating with linaclotide include, without limitation, talc,microcrystalline cellulose (e.g., AVICEL PH101 or CELPHERE CP-305),powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol,starch, pre-gelatinized starch, lactose, glucose, fructose, galactose,trehalose, sucrose, maltose, isomalt, dibasic calcium phosphate,raffinose, maltitol, melezitose, stachyose, lactitol, palatinite,xylitol, mannitol, myoinositol, and mixtures thereof.

As used herein, the term “additives” refers to any pharmaceuticallyacceptable additive. Pharmaceutically acceptable additives include,without limitation, disintegrants, dispersing additives, lubricants,glidants, antioxidants, coating additives, diluents, surfactants,flavoring additives, humectants, absorption promoting additives,controlled release additives, anti-caking additives, anti-microbialagents (e.g., preservatives), colorants, desiccants, plasticizers anddyes.

As used herein, an “excipient” is any pharmaceutically acceptableadditive, filler, binder or agent.

As used herein, “purified linaclotide” is linaclotide or apharmaceutically acceptable salt thereof that is greater than or equalto 90 percent pure or greater than or equal to 95 percent pure. In someembodiments, linaclotide as used in the methods and compositionsdescribed herein is purified. Linaclotide purity can be measured, forexample, by chromatographic purity of linaclotide using reversed phaseHPLC as described in Example 21. Linaclotide Assay [w/w] can bedetermined, for example, by using reversed phase HPLC with quantitationvia external calibration with a reference standard as described inExample 21.

In one aspect, the pharmaceutical composition may be prepared byspraying a solution comprising linaclotide or a pharmaceuticallyacceptable salt thereof, on a pharmaceutically acceptable filler togenerate linaclotide-coated filler. In one embodiment, the methodcomprises: (a) providing a solution, e.g., an aqueous solution (“thecoating solution”), comprising: (i) linaclotide or a pharmaceuticallyacceptable salt thereof; (ii) a cation selected from Mg²⁺, Ca²⁺, Zn²⁺,Mn²⁺, K⁺, Na⁺ or Al³⁺ and/or a sterically hindered primary amine (e.g.,leucine) and, optionally, (iii) a pharmaceutically acceptable binder;and (b) applying the coating solution to a pharmaceutically acceptablefiller to generate polypeptide-coated filler (e.g., by spraying, mixingor coating the pharmaceutically acceptable filler with the coatingsolution). The method can optionally include one or more of: (i)blending the polypeptide-coated filler with a pharmaceuticallyacceptable glidant, a pharmaceutically acceptable lubricant or apharmaceutically acceptable additive that acts as both a glidant andlubricant; (ii) blending the polypeptide-coated filler with filler thatis not polypeptide-coated, (iii) blending the polypeptide-coated fillerwith other additives; and (iv) applying a pharmaceutically acceptablecoating additive to the polypeptide-coated filler. The finalpharmaceutical composition can be placed into capsules (e.g., gelatincapsule) or used to form tablets.

In another embodiment, the pharmaceutical composition is prepared byspray drying, which is a technique used to prepare microparticles (e.g.,microcapsules or microspheres) of drugs. Spray-dried peptides generallyretain their biological activity upon dissolution and may have usefulphysical characteristics, including a uniform particle size and aspherical shape. In addition, the microparticles prepared by spraydrying are often free flowing, which is helpful for pharmaceuticalmanufacturing processes such as forming tablets and filling capsules.Spray drying processes are also useful because they may be readilyscaled up for clinical and commercial manufacturing.

Thus, this disclosure features a method for preparing a pharmaceuticalcomposition comprising linaclotide or a pharmaceutically acceptable saltthereof, the method comprising: (a) providing a solution, e.g., anaqueous or organic solution, comprising: (i) linaclotide or apharmaceutically acceptable salt thereof; and (ii) a cation selectedfrom Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ and/or a stericallyhindered primary amine (e.g., leucine) and (b) spray drying thelinaclotide-containing solution to produce microparticles. Thelinaclotide-containing solution can optionally include a polymer, suchas one or more of the binders described herein, a lipid or phospholipid,and/or a filler, such as mannitol. The method can optionally include oneor more additional steps of: (i) blending the linaclotide microparticleswith a pharmaceutically acceptable glidant, a pharmaceuticallyacceptable lubricant or a pharmaceutically acceptable additive that actsas both a glidant and lubricant; (ii) blending the microparticles with afiller, and/or (iii) blending the microparticles with other additives.The final pharmaceutical composition can be placed into capsules (e.g.,gelatin capsule) or used to form tablets.

In other embodiments, the pharmaceutical composition is prepared byspray freeze drying, supercritical fluid processing or lyophilization ofa solution, e.g., an aqueous or organic solution, comprising: (i)linaclotide or a pharmaceutically acceptable salt thereof; and (ii) acation selected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ and/or asterically hindered primary amine (e.g., leucine).

In some embodiments, the linaclotide composition is provided in a solidform for oral administration. Examples of such forms include, withoutlimitation, a tablet, a sachet, a pellet, a capsule or a powder. In someembodiments, the compositions can be used to create unit dosages forms,e.g., tablets, capsules, sachets or pellets. Orally administeredcompositions can include, for example, binders, lubricants, inertdiluents, lubricating, surface active or dispersing additives, flavoringadditives, and humectants. Orally administered formulations such astablets may optionally be coated or scored and may be formulated so asto provide sustained, delayed or controlled release of the linaclotidetherein. The linaclotide can be co-administered or co-formulated withother medications. In one embodiment, the linaclotide composition can beco-administered with other medications used to treat gastrointestinaldisorders. The linaclotide composition can also be used for treatment ofdisorders outside the gastrointestinal tract such as congestive heartfailure and benign prostatic hypertrophy.

The compositions can include, for example, various additional solvents,dispersants, coatings, absorption promoting additives, controlledrelease additives, and one or more inert additives (which include, forexample, starches, polyols, granulating additives, microcrystallinecellulose, diluents, lubricants, binders, disintegrating additives, andthe like), etc. If desired, tablet dosages of the disclosed compositionsmay be coated by standard aqueous or non-aqueous techniques.Compositions can also include, for example, anti-caking additives,preservatives, sweetening additives, colorants, flavors, desiccants,plasticizers, dyes, and the like.

Suitable disintegrants include, for example, agar-agar, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, povidone, polacrilin potassium, sodium starch glycolate,potato or tapioca starch, other starches, pre-gelatinized starch, clays,other algins, other celluloses, gums, and mixtures thereof.

Suitable lubricants include, for example, calcium stearate, magnesiumstearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol,polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate,talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zincstearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL200, W.R. Grace Co., Baltimore, Md. USA), a coagulated aerosol ofsynthetic silica (Evonik Degussa Co., Plano, Tex. USA), a pyrogenicsilicon dioxide (CAB-O-SIL, Cabot Co., Boston, Mass. USA), and mixturesthereof.

Suitable glidants include, for example, leucine, colloidal silicondioxide, magnesium trisilicate, powdered cellulose, starch, talc, andtribasic calcium phosphate.

Suitable anti-caking additives include, for example, calcium silicate,magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc,and mixtures thereof.

Suitable anti-microbial additives that may be used, e.g., as apreservative for the linaclotide compositions, include, for example,benzalkonium chloride, benzethonium chloride, benzoic acid, benzylalcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol,dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethylalcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate,potassium sorbate, propylparaben, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, andmixtures thereof.

Suitable coating additives include, for example, sodium carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, gelatin,pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methyl cellulose phthalate,methylcellulose, polyethylene glycol, polyvinyl acetate phthalate,shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax,and mixtures thereof. Suitable protective coatings include AQUACOAT®(e.g. AQUACOAT Ethylcellulose Aquaeous Dispersion, 15% w/w, FMCBiopolymer, ECD-30), EUDRAGIT (e.g. EUDRAGIT E PO PE-EL, Roehm PharmaPolymers) and OPADRY (e.g OPADRY AMB dispersion, 20% w/w, Colorcon).

In certain embodiments, suitable additives for the linaclotidecomposition include one or more of sucrose, talc, magnesium stearate,crospovidone or BHA.

In certain embodiments, the term “95%” may be 95.0%, the term “90%” maybe 90.0%, the term “10%” may be 10.0%, the term “9%” may be 9.0%, theterm “8%” may be 8.0%, the term “7%” may be 7.0%, the term “6%” may be6.0%, the term “5%” may be 5.0%, the term “4%” may be 4.0%, the term“3%” may be 3.0%, the term “2%” may be 2.0%, and the term “1%” may be1.0%.

In certain embodiments, the linaclotide composition is provided in aunit dosage form. In some embodiments, the unit dosage form is acapsule, a tablet, a sachet, a pellet or a powder. In one suchembodiment, the unit dosage form is a capsule or tablet. Such unitdosage forms may be contained in a container such as, withoutlimitation, a paper or cardboard box, a glass or plastic bottle or jar,a re-sealable bag (for example, to hold a “refill” of tablets forplacement into a different container), or a blister pack with individualdoses for pressing out of the pack according to a therapeutic schedule.It is feasible that more than one container can be used together in asingle package to provide a single dosage form. For example, tablets orcapsules may be contained in a bottle which is in turn contained withina box. In some embodiments, the unit dosage forms are provided in acontainer further comprising a desiccant. In a further embodiment, theunit dosage forms, e.g., a quantity of tablets or capsules, are providedin a container, e.g., a bottle, jar or re-sealable bag, containing adesiccant. In a further embodiment, the container containing the unitdosage forms is packaged with administration or dosage instructions. Incertain embodiments, the linaclotide composition is provided in a kit.The linaclotide composition described herein and combination therapyagents can be packaged as a kit that includes single or multiple dosesof two or more agents, each packaged or formulated individually, orsingle or multiple doses of two or more agents packaged or formulated incombination. Thus, the linaclotide composition can be present in firstcontainer, and the kit can optionally include one or more agents in asecond container. The container or containers are placed within apackage, and the package can optionally include administration or dosageinstructions.

EXAMPLES

The following examples are merely illustrative of the present inventionand should not be construed as limiting the scope of the invention inany way as many variations and equivalents that are encompassed by thepresent invention will become apparent to those skilled in the art uponreading the present disclosure.

Linaclotide or a pharmaceutically acceptable salt thereof may beproduced and purified using standard techniques known in the art, e.g.,chemical synthesis or recombinant expression followed by andpurification using standard techniques.

Formulation Scheme A

Preparation of the Coating Solution:

Approximately 32 g to 42 g of purified water is mixed with hydrochloricacid to create a solution with a pH between 1.5 and 2.0. The cation, ifused, is added to the solution in a quantity to provide the desiredconcentration, and the solution is mixed for sufficient time to producea clear solution. The sterically hindered primary amine, if used, isadded to the solution in a quantity to provide the desiredconcentration, and the solution is mixed for sufficient time to producea clear solution. Other additives, such as antioxidants, are then added,if desired. The pH of the solution is tested, and hydrochloric acid isadded, if necessary, to produce a solution having a pH between 1.5 and2.0. The binder is then added to the solution and the mixture is thenstirred for sufficient time to achieve a clear solution. The desiredamount of linaclotide is added to the solution and mixed for 30-100minutes to provide the coating solution.

Preparation of the Active Beads:

Approximately 30-36 g of dried microcrystalline cellulose beads areadded to a Mini Column Fluid Bed Coater. The microcrystalline cellulosebeads are fluidized and heated prior to layering. Next, the coatingsolution is layered to the beads. The spraying temperature is controlledbetween 24° C. and 55° C. by controlling inlet temperature, spray rate,atomization pressure, and air volume. After the entire coating solutionis layered to the beads, the beads are dried. The product of thisprocess is referred to as active beads.

Preparation of Active Beads with Protective Coating: Approximately 35 gof Active Beads are added to a Mini Column Fluid Bed Coater. The ActiveBeads are fluidized and heated prior to coating with AQUACOAT (e.g.Aquacoat Ethylcellulose Aquaeous Dispersion, 15% w/w, FMC Biopolymer,ECD-30), EUDRAGIT (e.g. EUDRAGIT E PO PE-EL, Roehm Pharma Polymers) orOPADRY (e.g Opadry AMB dispersion, 20% w/w, Colorcon). Next, the coatingsolution is layered to the beads. The spraying temperature is controlledbetween 24° C. and 55° C. by controlling inlet temperature, spray rate,atomization pressure, and air volume. After the entire coating solutionis layered to the beads, the beads are dried.

Formulation Scheme B

Preparation of the Coating Solution:

Approximately 8.3 kg of purified water is mixed with hydrochloric acidto create a solution with a pH between 1.5 and 2.0. The cation, if used,is added to the solution in a quantity to provide the desiredconcentration, and the solution is mixed for sufficient time to producea clear solution. The sterically hindered primary amine, if used, isadded to the solution in a quantity to provide the desiredconcentration, and the solution is mixed for sufficient time to producea clear solution. Other additives, such as antioxidants, are then added,if desired. The binder is then added to the solution and the solution ismixed for sufficient time to achieve a clear solution. The pH of thesolution is tested, and hydrochloric acid is added if necessary toproduce a solution having a pH between 1.5 and 2.0. This is Solution 1.Approximately 8.3 kg of purified water is mixed with hydrochloric acidto create a solution with a pH between 1.5 and 2.0. The desired amountof linaclotide is added to the solution and mixed for 10 to 30 minutes.The pH of the solution is tested, and hydrochloric acid is added ifnecessary to produce a solution having a pH between 1.5 and 2.0. This isSolution 2. Solution 1 and Solution 2 are then mixed together. The pH ofthe solution is tested, and hydrochloric acid is added if necessary toproduce a solution having a pH between 1.5 and 2.0. This is the coatingsolution.

Preparation of the Active Beads:

Approximately 24.19 kg of microcrystalline cellulose beads are added toa Wurster Column of a Glatt GPCG-30 Fluid Bed. The microcrystallinecellulose beads are fluidized and heated to product temperature of45-47° C. Next, the coating solution is layered to the beads. Theproduct spraying temperature is controlled between 37° C. and 47° C. bycontrolling inlet temperature, spray rate, atomization pressure, and airvolume. After the entire coating solution is layered to the beads, thebeads are dried with a product drying temperature of 37° C. to 47° C.The product of this process is referred to as active beads.

Examples 1-15 Preparation of Linaclotide Formulations

The linaclotide formulations of Examples 1-15 were produced essentiallyas described in Formulation Scheme A wherein Table 1 provides theamounts of cation, sterically hindered primary amine, binder,linaclotide and beads, while Table 2 provides the conditions under whichthe beads were coated:

TABLE 1 Cation Amine Amount Amount Binder Amount of Beads Example [ ]* [] Amount Linaclotide ** Amount  1 CaCl₂•2H₂O Leucine Hypromellose 0.1282g Celphere CP-305 0.6740 g 0.2005 g 1.019 g 33.38 g [60] [20]  2CaCl₂•2H₂O Leucine Hypromellose 0.1329 g Celphere CP-305 0.6740 g 0.3007g 0.3063 g 33.87 g [60] [30]  3 CaCl₂•2H₂O Leucine Hypromellose 01282 gCelphere CP-305 0.2247 g 1.002 g 0.0656 g 33.86 g [20] [100]  4CaCl₂•2H₂O Leucine Hypromellose 0.1282 g Celphere CP-305 1.123 g 0.2005g 1.969 g 32.36 g [100] [20]  5 CaCl₂•2H₂O Leucine Hypromellose 0.1282 gCelphere CP-305 0.4493 g 0.4009 g 0.5425 g 33.78 g [40] [40]  6MgCl₂•6H₂O Leucine Hypromellose 0.2100 g Celphere CP-305 0.2590 g 0.3341g 0.6636 g 33.83 g [10] [20]  7 ZnAc•2H₂O Leucine Hypromellose 0.2100 gCelphere CP-305 0.2796 g 0.3341 g 0.6636 g 33.82 g [10] [20]  8 N/ALeucine Hypromellose 0.4387 g Celphere CP-305 0.8944 g 0.6636 g 33.40 g[27]  9 CaCl₂•2H₂O N/A Hypromellose 0.4227 g Celphere CP-305 0.3745 g0.6636 g 33.83 g [10] 10 N/A N/A Hypromellose 0.2114 g Celphere CP-3050.6811 g 34.28 g 11 N/A N/A Hypromellose 0.4227 g Celphere CP-305 0.6636g 34.13 g 12 CuCl₂•2H₂O N/A Hypromellose 0.4227 g Celphere CP-305 0.4342g 0.6636 g 33.79 g [10] 13 ZnAc•2H₂O N/A Hypromellose 0.4227 g CelphereCP-305 0.5590 g 0.6636 g 33.68 g [10] 14 MgCl₂•6H₂O N/A Hypromellose0.4227 g Celphere CP-305 0.5178 g 0.6636 g 33.72 g [10] 15 N/AMethionine Hypromellose 0.4387 g Celphere CP-305 0.0380 g 0.6636 g 34.08g [1] *“Cation” refers to the divalent cation contained in the salt usedin the example, “Amine” refers to the sterically hindered primary amine,[ ] refers to the molar ratio of the cation and/or amine to linaclotide.** The Amount of linaclotide in this and all following examples isdetermined based on peptide content and chromatographic purity as listedon the Certificate of Analysis provided for each manufactured lot oflinaclotide Active Pharmaceutical Ingredient (API).

TABLE 2 Product Inlet Atomization Spraying Temp Spray rate Pressure AirExample Temp (° C.) (° C.) (mL/min) (psig) Flow  1 34.0-37.0 55.7-57.70.33-0.40 20 Low  2 27.4-32.3 37.01-42.1  0.40 22 Low  3 32.6-34.760.0-60.1 0.33-0.40 20 Low  4 35.3-39.3 58.9-59.2 0.40 18 Low  527.8-27.9 58.7-59.8 0.35-0.33 20 Low  6 32.1-38.3 42.0-53.4 0.39-0.75 22Low  7 31.7-39.3 50.0-52.5 0.27-0.57 22 Low  8 33.3-41.3 50.5-57.00.57-0.65 22 Low  9 33.2-40.0 49.5-58.7 0.82-1.00 20 Low 10 42.5 59.50.49 22 Low 11 39.7 52.0 0.66 22 Low 12 36.6-40.0 47.2-54.8 0.65-0.7520-22 Low 13 32.4 57.4 0.65 22 Low 14 34.0 49.0 0.75 20 Low 15 24.1-39.948.5-55.9 0.39-0.65 22-23 Low

Example 16 Preparation of Linaclotide Formulation

The linaclotide formulation of Example 16 was produced essentially asdescribed in Formulation Scheme B wherein Table 3 provides the amountsof cation, sterically hindered primary amine, binder, linaclotide andbeads, while Table 4 provides the conditions under which the beads werecoated:

TABLE 3 Cation Amine Amount Amount Amount Binder of Beads Example [ ] [] Amount Linaclotide Amount 16 CaCl₂•2H₂O Leucine Hypro- 73.5 g Celphere385.1 g 171.8 g mellose CP-305 [60] [30] 175.0 g 24.19 kg

TABLE 4 Atom- Process Product Product Inlet Spray ization Air DryingSpraying Temp rate Pressure Volume Temp Example Temp (° C.) (° C.)(g/min) (bar) (cfm) (° C.) 16 64.9-65.1 80 150 2.0 515-564 54.9-55.0

Example 17 Preparation of Linaclotide Formulation

The linaclotide formulation of Example 17 was produced essentially asdescribed in Formulation Scheme A except that the formulation contained22.96 mg butylated hydroxyanisole (BHA), wherein Table 5 provides theamounts of cation, sterically hindered primary amine, binder,linaclotide and beads, while Table 6 provides the conditions under whichthe beads were coated.

TABLE 5 Cation Amine Amount Amount Amount Binder of Beads Example [ ] [] Amount Linaclotide Amount 17 CaCl₂•2H₂O N/A Hypro- 0.2100 g Celphere0.3745 g mellose CP-305 [20] 0.6636 g 33.99 g

TABLE 6 Product Inlet Atomization Spraying Temp Spray rate Pressure AirExample Temp (° C.) (° C.) (mL/min) (psig) Flow 17 33.5-34.8 47.7-48.60.56-0.74 26 Low

Example 18 Preparation of Capsules Containing Linaclotide Formulation

The linaclotide content on active beads may be measured as described inExample 21 or by other equivalent methods.

To form capsules suitable for oral administration, an appropriate amountof active beads is used to fill gelatin capsules (e.g., Size 2 gelatincapsules). An appropriate amount of active beads may contain 50 μg to 2mg linaclotide per capsule with a range of ±5%. In some embodiments, theappropriate amount of linaclotide on active beads may be 50 μg, 67.5 μg,100 μg, 133 μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg, 600 μg,700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 4 mg or 6 mg. In a particularembodiment, the appropriate amount of linaclotide on active beads is67.5 μg, 100 μg, 133 μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg,600 μg. In a more particular embodiment, the appropriate amount oflinaclotide on active beads is 67.5 μg, 133 μg, 150 μg, 266 μg or 300 μgper capsule.

In another embodiment, an appropriate amount of active beads to fill adesired number of gelatin capsules is placed in a container. One or morepharmaceutically acceptable fillers or other pharmaceutically acceptableadditives may be added, if desired, to the container. In someembodiments, a filler or additive is talc, leucine, microcrystallinecellulose or mannitol. The contents of the container are blended and themixture is used to fill gelatin capsules with an appropriate amount ofactive beads containing linaclotide (e.g., 50 μg to 2 mg linaclotide percapsule with a range of ±5%).

In an alternative embodiment, an appropriate amount of active beads isused to fill gelatin capsules and one or more pharmaceuticallyacceptable fillers or other pharmaceutically acceptable additives areadded to the gelatin capsules.

Example 19 Preparation of Capsules Containing Linaclotide Formulation

Preparation of the Coating Solution: First, 41.98 g of purified waterwas mixed with 1.13 g of hydrochloric acid in order to create a solutionwith a pH between 1.5 and 2.0. Next, 7.49 g of calcium chloridedihydrate and 6.68 g of leucine were added to the solution, which wasthen mixed for 30 minutes in order to produce a clear solution. The pHwas tested, and 1.70 g of hydrochloric acid was added to produce asolution having a pH between 1.5 and 2.0. Next, 13.27 g of hypromellose(hydroxypropyl methylcellulose; Dow Chemical Company; Midland, Mich.)was added to the solution and the mixture was stirred for 60 minutes toachieve a clear solution. Next, 4.39 g of a linaclotide was added to thesolution and mixed for 90 minutes. The pH of the solution was 1.73. Thiswas the coating solution.

Preparation of the Active Beads: 674.5 g of microcrystalline cellulosebeads (Celphere CP-305; Ashai Kasei Corporation (Tokyo; Japan) wereadded to a Wurster Column of a Glatt GPCG-2 Fluid Bed. Themicrocrystalline cellulose beads were fluidized and heated for 30minutes at a product temperature of 60° C. Next, the coating solutionwas layered to the beads. The product temperature was controlled between45° C. and 49° C. by an inlet temperature of 80° C., spray rate of5.0-11 g/min, an atomization pressure of 2.0 bar, and air volume of 40to 50 m³h. After the entire coating solution was layered to the beads,the beads were dried for 10 minutes with a product temperature of 46.9°C. to 50.9° C. The product of this process was referred to as activebeads.

Reverse phase liquid chromatography of linaclotide extracted from aformulation prepared as described above demonstrated that the extractedlinaclotide and a linaclotide reference standard exhibited the sameretention time and that there was no significant change in purity as aresult of the formulation process.

To form capsules, 49.50 g of active beads were added to a clear bag.Next, 0.25 g of leucine, screened through a 60 mesh screen, was added tothe bag. The bag was tied and mixed for 125 turns in order to blend allof the materials. Next, 0.25 g of talc, screened through a 60 meshscreen, was added to the bag. The bag was tied and mixed for 125 turnsto blend all of the materials. Once all of the materials were blended,the mixture was used to fill Size 2 gelatin capsules at target weight of227 mg/capsule with a range of ±5%.

Example 20 Preparation of Capsules Containing Linaclotide Formulation

Active beads were prepared according to Example 16. The active beadswere tested for linaclotide content. Based on the assay of the activebeads, an appropriate amount of active beads (96 mg-123 mg) were filledinto size 2 hard gelatin capsules using an MG2 Futura encapsulationmachine, to achieve a linaclotide concentration of 300 μg.

Active beads were prepared according to Example 15. The active beadswere tested for linaclotide content. Based on the assay of the activebeads, an appropriate amount of active beads (48 mg-62 mg) were filledinto size 2 hard gelatin capsules using an MG2 Futura encapsulationmachine, to achieve a linaclotide concentration of 150 μg.

Example 21 Measurement of Linaclotide Content and Purity

Linaclotide content and purity, as well as measurement oflinaclotide-related substances may be determined by reverse phasegradient liquid chromatography using an Agilent Series 1100 LC Systemwith Chemstation Rev A.09.03 software or equivalent. A YMC Pro™ C18column (dimensions: 3.0×150 mm, 3.5 um, 120 Å; Waters Corp., Milford,Mass.) or equivalent is used and is maintained at 40° C. Mobile phase A(MPA) consists of water with 0.1% trifluoroacetic acid while mobilephase B (MPB) consists of 95% acetonitrile:5% water with 0.1%trifluoroacetic acid. Elution of linaclotide and its related substancesis accomplished with a gradient from 0% to 47% MPB in 28 minutesfollowed by a ramp to 100% MPB in 4 minutes with a 5 minute hold at 100%MPB to wash the column. Re-equilibration of the column is performed byreturning to 0% MPB in 1 minute followed by a 10 minute hold at 100%MPA. The flow rate is 0.6 mL/min and detection is accomplished by UV at220 nm.

Samples for analysis are prepared by addition of the contents oflinaclotide capsules to 0.1 N HCl to obtain a target concentration of 20μg linaclotide/mL. 100 μL of this solution is injected onto the column.

Linaclotide content is measured by determining the linaclotideconcentration in the prepared sample against a similarly preparedexternal linaclotide standard.

An example of an analysis of linaclotide by HPLC is shown in FIG. 1,wherein “Oxidation” refers to the linaclotide oxidation product,“Formaldehyde Imine” refers to the linaclotide formaldehyde imineproduct and “Hydrolysis” refers to the linaclotide hydrolysis product.

Example 22 Linaclotide Formulation Stability Testing

For the formulations of Examples 1-15 and 17, gelatin capsules werefilled with approximately 225 mg of active beads. Five filled capsuleswere placed in plastic bottles. The bottles contained 1 to 2 g ofdesiccant and were induction sealed. The bottles were stored at 40°C./75% RH for six months.

Linaclotide content and purity as well as the amount oflinaclotide-related substances were measured essentially as described inExample 21 or by an equivalent method. Results are provided in Table 7.

TABLE 7 Assay Area % by HPLC [w/w] % Linaclotide Formaldehyde Example ofInitial (% of Initial) Oxidation Hydrolysis Imine  1 107.56 96.88 0.110.24 0.19 (99.13)  3  98.87 97.36 0.07 0.52 0.15 (99.42)  4  95.67 95.610.10 0.16 0.24 (97.83)  5 103.41 95.87 0.07 0.25 0.24 (98.68)  6  99.4693.64 0.14 0.70 0.55 (95.51)  7  98.64 93.44 0.45 1.45 0.63 (95.36)  8 92.81 88.20 0.37 1.85 0.49 (94.90)  9  93.53 93.81 0.2 0.41 1.06(96.55) 10  77.12 84.85 0.37 0.29 4.45 (87.77) 11  85.73 89.09 1.18 0.491.38 (91.63) 12  33.60 41.98 ND ND ND (43.15) 13  87.69 91.91 1.98 0.740.86 (94.01) 14  86.94 90.59 0.25 0.54 1.23 (92.70) 15  87.71 87.54 0.240.66 1.67 (93.24) 17  98.94 93.65 ND 0.32 0.73 (95.16)

For the formulation of Example 16, gelatin capsules were filled withapproximately 113 mg of total beads. 35 filled capsules were placed inplastic bottles. The bottles contained 2 g of desiccant and wereinduction sealed. The bottles were stored at 40° C./75% RH for onemonth.

Linaclotide content and purity as well as the amount oflinaclotide-related substances may be measured essentially as describedin Example 21 or by an equivalent method. Results are shown in Table 8.

TABLE 8 Assay Area % by HPLC [w/w] % Linaclotide Ox- Hy- FormaldehydeExample of Initial (% of Initial) idation drolysis Imine 16 97.01 97.12<0.1 <0.1 0.34 (99.79)

Example 23 Isolation and Preparation of Linaclotide Hydrolysis Product

The linaclotide hydrolysis product occurs as a transformation of Asn inthe 7 position to Asp (the numbering of linaclotide starts with 1 at theN-terminal Cys). Its structure is depicted below:

The linaclotide hydrolysis product has been independently synthesizedfor confirmation of identity using standard solid phase peptidesynthesis techniques. The linaclotide hydrolysis product may also beprepared by other methods known in the art, e.g., by isolation fromlinaclotide preparations using chromatographic techniques or byrecombinant expression of a nucleic acid encoding the linaclotidehydrolysis product (Cys Cys Glu Tyr Cys Cys Asp Pro Ala Cys Thr Gly CysTyr; SEQ ID NO: 2), optionally followed by oxidation of the cysteineresidues to form the disulfide linkages.

Example 24 Isolation and Preparation of Linaclotide Formaldehyde ImineProduct

The formaldehyde imine product occurs as the addition of an imine to theN-terminal Cys (Cys1) via a formaldehyde-mediated reaction. A proposedstructure of the product is depicted below:

The linaclotide formaldehyde imine product has been independentlysynthesized for confirmation of identity by reacting linaclotide withformaldehyde (1:5 molar ratio) in absolute ethanol at room temperaturefor 4 days. The formaldehyde imine product may also be prepared by othermethods known in the art, e.g., by isolation from linaclotidepreparations using chromatographic techniques or by chemical peptidesynthesis or recombinant expression of a nucleic acid encodinglinaclotide followed by formylation as described herein or by othermethods known in the art, optionally followed by oxidation of thecysteine residues to form the disulfide linkages.

Example 25 Isolation and Preparation of Linaclotide Oxidation Product

The linaclotide oxidation product has a molecular weight of 1542.8. Theoxidation product most likely forms as the addition of a single oxygenatom to one of the six cysteinyl sulfurs in linaclotide. One potentialstructure of the product is depicted below, although one of skill in theart will recognize that the oxygen atom may be attached to any of theother five sulfurs:

To support this identification, the linaclotide oxidation product hasbeen produced by reacting linaclotide with hydrogen peroxide (3%aqueous) at room temperature or 40° C. for up to 24 hours. The resultingproduct is enriched in the oxidation product by 1-10%. The linaclotideoxidation product may also be prepared by other methods known in theart, e.g., by isolation from linaclotide preparations usingchromatographic techniques or by chemical peptide synthesis orrecombinant expression of a nucleic acid encoding linaclotide followedby oxidation of the cysteine residues to form the disulfide linkagesfollowed by reacting linaclotide with hydrogen peroxide or similaroxidizing reagent to form the linaclotide oxidation product.

Example 26 Linaclotide Tablet Formation Fluid Bed Granulation

Linaclotide, CaCl₂, leucine and polyvinyl pyrrolidone (PVP) K30 weredissolved in 0.0001N HCl to form the coating solution (see Table 9).Isomalt was charged to the bowl of the fluid bed. With fluidizing theisomalt powder, the drug solution was top-sprayed at a speed of ˜10g/min, with product temperature of ˜40° C. to coat the powder with thecoating solution. Upon finishing spraying, the linaclotide granules weredried for 30 minutes and the product was discharged.

TABLE 9 Cation Amine Exam- Amount Amount Binder Amount of Filler ple [ ][ ] Amount Linaclotide Amount 26A CaCl₂•2H₂O Leucine PVP K30 3.08 gIsomalt 15.4 g 6.9 g 40 g 935 g [60] [30]

Dicalcium phosphate or Avicel were also used as filler for fluid bedgranulation.

Wet Granulation

Linaclotide was weighed and dissolved under agitation in 250 g of 0.1 NHCl (pH 1.7) to form Solution 1 (see Table 10). CaCl₂ and leucine wereweighed and dissolved under agitation in 100 g 0.1 N HCl to formSolution 2. Solution 1 and Solution 2 were mixed together underagitation to form the coating solution. Avicel was added to the bowl ofa high shear granulator. With mixing at 500 rpm, the coating solutionwas added into the Avicel. Upon finishing adding the solution, thegranules were mixed and chopped for 1 minute. The wet granules obtainedwere charged into the bowl of a fluid bed, and dried for 15 minute andthen the linaclotide granules were discharged.

TABLE 10 Cation Amine Exam- Amount Amount Binder Amount of Filler ple [] [ ] Amount Linaclotide Amount 26B CaCl₂•2H₂O Leucine N/A 1.54 gIsomalt 7.68 g 3.42 g 488 g [60] [30]

In the wet granulation formula, the molar ratio of CaCl₂ and leucine tolinaclotide was adjusted in the range of 60 to 100 and 30 to 50,respectively. Also, sucrose was added in one example. See Table 11.

TABLE 11 Strength Exam- (Linaclotide/ Su- ple Filler) FillerCaCl₂:Leu:Linaclotide crose HCl 26C 600 μg/225 mg Avicel 60:30:1 No 0.1N26D 600 μg/225 mg Avicel 80:40:1 No 0.1N 26E 600 μg/225 mg Avicel100:50:1  No 0.1N 26F 600 μg/225 mg Avicel 60:30:1 5% 0.1N

Tablet Formulation

The linaclotide granules were blended with the following excipients (seeTable 12) and compressed into tablets with a hardness of ˜4 kp.

TABLE 12 Weight in Weight in Weight in Weight in 200 mg tablet 400 mgtablet 800 mg tablet 1600 mg tablet Ingredient with 150 μg with 300 μgwith 600 μg with 1200 μg Function Linaclotide Linaclotide LinaclotideLinaclotide Linaclotide 53.4 mg 106.8 mg 213.6 mg 427.2 mg granules APIIsomalt 134.1 mg 268.2 mg 536.4 mg 1072.8 mg Tablet filler Crospovidone10 mg 20 mg 40 mg 80 mg Disintegrate Magnesium 1.5 mg 3 mg 6 mg 12 mgstearate Lubricant Talc Glidant 1 mg 2 mg 4 mg 8 mg Total of dry 200 mg400 mg 800 mg 1600 mg material

Isomalt, starch 1500 or dicalcium phosphate were also used as the tabletfiller based on the above formula (see Table 13).

TABLE 13 Granulation Filler CaCl₂:leucine:Linaclotide Tablet FillerFluid bed isomalt 60:30:1 isomalt starch 1500 dicalcium phosphate Fluidbed Avicel 60:30:1 starch 1500 Wet Avicel 100:50:1  starch 1500granulation Wet Avicel 60:30:1 + 5% sucrose starch 1500 granulation

After two weeks storage at 40° C. and 75% relative humidity, all tabletsdescribed in Table 13 exhibited assay values of linaclotide is greaterthan 90%.

Examples 27-53 Preparation of Linaclotide Formulations

The linaclotide formulations of Examples 27-53 were produced essentiallyas described in Formulation Scheme A and Examples 1-15. The linaclotidecoating solution contained 0.7% binder (w/v) and the coating solutionwas sprayed on CELPHERE CP-305 beads as described in Examples 1-15.Table 14 provides the type of cation, amine and/or other excipient alongwith their molar ratios relative to linaclotide, as well as the type ofbinder used, while Table 15 provides the conditions under which thebeads were coated:

TABLE 14 Example Cation Amine Molar Ratio Binder Additive 27 CaCl₂•2H₂O— 20:0:1 Hypromellose — 28 MnCl₂•4H₂O — 20:0:1 Hypromellose — 29 KCl —20:0:1 Hypromellose — 30 AlCl₃•6H₂O — 20:0:1 Hypromellose — 31CaCl₂•2H₂O Leucine 60:30:1 Hypromellose — 32 Ca Alginate Leucine 60:30:1Hypromellose — 33 CaHPO₄ Leucine 60:30:1 Hypromellose — 34 Ca StearateLeucine 60:30:1 Hypromellose — 35 CaSO₄•2H₂O Leucine 60:30:1Hypromellose — 36 Zn(OAc)₂ Leucine 60:30:1 Hypromellose — 37 CaCl₂•2H₂OIsoleucine 60:30:1 Hypromellose — 38 CaCl₂•2H₂O Valine 60:30:1Hypromellose — 39 CaCl₂•2H₂O Methionine 60:30:1 Hypromellose — 40CaCl₂•2H₂O Phenylalanine 60:30:1 Hypromellose — 41 — Histidine  0:20:1Hypromellose — 42 — Tryptophan  0:20:1 Hypromellose — 43 CaCl₂•2H₂O —0:20:1:20 Hypromellose Vitamin E (Vit. E) 44 — 1-aminocyclohexane 0:20:1 Hypromellose — carboxylic acid 45 — cyclohexylamine  0:20:1Hypromellose — 46 — 2-methylbutylamine  0:20:1 Hypromellose — 47 —chitosan  0:20:1 Hypromellose — 48 CaCl₂•2H₂O Leucine 60:30:1 Polyvinyl— pyrrolidone 49 CaCl₂•2H₂O Leucine 60:30:1 Methyl cellulose — (MethocelA15) 50 CaCl₂•2H₂O Leucine 60:30:1 Hydroxypropyl — cellulose 51 NaCl —20:0:1 Hypromellose — 52 CaCl₂•2H₂O Leucine 60:30:1 Gelatin — 53CaCl₂•2H₂O Glycine 60:30:1 Hypromellose — * “Cation” refers to thecation contained in the salt used in the example, “Amine” refers to thesterically hindered primary amine, “Molar Ratio” refers to the molarratio of the cation:amine:linaclotide:Additive (if applicable).

TABLE 15 Product Spraying Inlet Spray Atomization Exam- Temp Temp ratePressure Air ple (° C.) (° C.) (g/min) (psig) Flow 27 25.1-35.137.0-50.1 0.44-0.62 20 Low 28 24.1-35.8 37.3-50.9 0.30-0.61 18-20 Low 2928.1-34.7 37.6-47.8 0.50-0.63 18 Low 30 29.8-35.0 33.9-50.2 0.32-0.47 20Low 31 25.5-35.1 34.6-50.4 0.40-0.61 20 Low 33 30.4-35.2 38.7-51.00.48-0.52 20 Low 35 29.9-34.9 37.8-50.4 0.37-0.76 20 Low 36 29.9-35.438.0-50.1 0.38-0.50 21 Low 37 27.3-34.9 36.2-50.1 0.45-0.54 20 Low 3827.6-36.2 36.9-47.3 0.43-0.66 20 Low 39 30.1-35.8 40.6-47.1 0.30-0.48 20Low 40 31.7-37.5 41.3-51.0 0.40-0.67 18 Low 41 29.4-36.2 41.7-49.50.48-0.53 20 Low 42 31.0-38.6 42.4-51.2 0.52-0.64 20 Low 44 31.0-37.639.5-48.8 0.40-0.46 18 Low 45 28.7-36.5 37.1-49.2 0.49-0.61 18 Low 4628.6-35.2 37.1-47.2 0.39-0.53 18 Low 47 33.4-38.7 40.6-48.5 0.48-0.4718-26 Low 48 31.6-36.1 41.6-46.7 0.36-0.72 18 Low 49 28.5-36.5 36.8-48.10.45-0.51 18 Low 50 27.9-36.4 37.1-48.6 0.35-0.60 18 Low 51 29.3-37.936.7-49.2 0.42-0.55 18 Low 52 29.8-36.3 36.1-49.1 0.44-0.54 18 Low 5328.9-35.8 36.5-47.7 0.45-0.52 18 Low

Processing issues were experienced during spraying on the beads forexamples 32 (Calcium Alginate), 34 (Calcium Stearate) and 43(CaCl₂:Vitamin E). Thus, the coating solution was mixed with theCELPHERE beads and the beads were dried on a tray.

Example 54 Linaclotide Formulation Stability Testing

For the formulations of Examples 27-53, gelatin capsules were filledwith approximately 225 mg of active beads (600 μg linaclotide/capsule).Five filled capsules were placed in plastic bottles. The bottlescontained 1 g of desiccant and were induction sealed. The bottles werestored at 40° C./75% RH for three months or six months.

Linaclotide content (μg/mg) and percent chromatographic purity (% CP)were measured essentially as described in Example 21 or by an equivalentmethod. Results are provided in Table 16A (three months stability) orTable 16B (six month stability).

TABLE 16A Exam- Assay [w/w] % CP ple % of Initial * % CP [% of Initial]27 96.30 93.98% 98.07 28 96.82 93.59% 96.07 29 101.56 92.71% 95.40 30109.06 93.07% 95.76 31 103.59 95.98% 99.12 32 66.53 82.66% 85.27 3396.81 91.94% 93.55 34 30.75 55.47% 56.88 35 101.37 93.07% 95.02 36105.27 91.49% 93.45 37 109.22 95.73% 97.99 38 99.24 95.79% 97.59 3995.22 95.76% 97.82 40 102.98 95.68% 97.60 41 110.92 94.03% 96.30 42120.05 88.57% 91.65 43 58.51 70.99% 74.06 44 98.83 93.84% 96.88 45 91.7290.07% 93.71 46 90.17 89.45% 91.67 47 105.70 88.59% 91.31 48 106.9295.11% 97.62 49 96.48 94.62% 96.60 50 112.30 95.86% 98.98 51 102.9291.80% 99.79 52 108.12 83.10% 86.80 53 104.22 95.25% 97.95 * Variabilityin the values for Assay [w/w % of Initial] reflects the imperfectcontrol over content uniformity for these capsule lots, whichmanufactured at small scale.

It is believed that the difficulties encountered during processing andthe resulting modified processing procedure for Examples 32, 34 and 43(see above) could explain the lower stability observed in these samples.

TABLE 16B Assay Area % by HPLC Exam- [w/w] % Linaclotide Oxida- Hydro-Formaldehyde ple of Initial (% of Initial) tion lysis Imine 27 91.5889.68 0.09 0.60 1.59 (93.58) 28 93.36 88.44 0.24 0.41 1.55 (90.78) 2993.73 87.79 0.18 0.53 1.82 (90.34) 30 108.63 93.93 0.39 1.11 0.44(96.65) 31 94.53 86.83 — 0.41 0.98 (89.67) 32 69.28 73.15 0.97 1.93 1.69(75.46) 33 88.91 85.96 0.97 3.86 0.17 (87.46) 34 77.37 70.42 0.67 0.991.78 (72.21) 35 95.34 88.85 0.39 1.80 0.33 (90.71) 36 102.83 87.27 3.311.86 0.21 (89.14) 37 99.33 87.23 — 0.59 0.25 (89.29) 38 93.97 86.27 —0.42 0.45 (87.89) 39 87.78 85.23 — 0.40 0.31 (87.07) 40 94.36 86.28 —0.46 0.41 (88.01) 41 104.28 90.04 0.33 1.61 0.52 (92.22) 42 117.92 76.850.14 1.21 0.10 (79.52) 43 54.21 59.54 5.92 4.44 1.83 (62.12) 44 92.5690.24 0.16 1.47 0.54 (93.17) 45 76.23 79.57 0.17 0.87 1.22 (82.78) 4673.07 78.92 0.51 0.66 0.65 (80.88) 47 97.65 82.73 0.92 0.60 2.68 (85.27)48 93.94 85.24 0.05 0.69 0.20 (87.49) 49 51.65 63.46 0.96 0.58 2.24(64.79) 50 104.75 92.61 — 0.38 0.48 (95.62) 51 94.15 88.19 — 0.58 1.35(92.01) 52 100.06 72.81 0.06 0.49 0.41 (75.62) 53 95.74 89.80 0.06 0.361.40 (92.35)

Chromatographic purity values for Examples 27-53 at the six-month timepoint appear atypically low, particularly with respect to thethree-month time points for these samples. Relative trends forstabilizing or destabilizing effects can be established by comparisonwith Example 27 and Example 31 as internal reference experiments, forwhich the chromatographic purity values are approximately 6-8% lowerthan consistently observed in other studies that have been conducted(see, e.g., Examples 2 and 9). The three month data provided in Table16A for the same formulations shows more typical chromatographic purityvalues. Thus, the low chromatographic purity values at six months arelikely due to an insufficient desiccant capacity at six months for theseparticular storage conditions. This hypothesis is supported by theimpurity peaks that are observed and that are indicative of exposure tomoisture.

Example 55 Linaclotide Formulation Stability Testing at 25° C./60% RHfor 24 Months

For the formulations of Examples 8-15 and 17, gelatin capsules werefilled with approximately 225 mg of active beads. Five filled capsuleswere placed in plastic bottles. The bottles contained 1 g of desiccantand were induction sealed. The bottles were stored at 25° C./60% RH for24 months.

Linaclotide content and purity as well as the amount oflinaclotide-related substances were measured essentially as described inExample 21 or by an equivalent method. Results are provided in Table 17.

TABLE 17 Assay Area % by HPLC Exam- [w/w] % Linaclotide Oxida- Hydro-Formaldehyde ple of Initial (% of Initial) tion lysis Imine  8 94.3694.58 0.21 1.26 0.53 (101.7)  9 94.08 95.09 0.14 0.36 0.93 (97.86) 1080.80 87.82 0.38 0.26 3.77 (90.84) 10a ¹⁾ 89.29 91.55 0.50 0.39 1.60(94.95 10b ²⁾ 88.41 91.19 0.44 0.34 1.61 (95.02) 10c ³⁾ 72.35 72.36 0.300.26 19.13 (75.76) 11 87.50 90.25 1.03 0.42 1.94 (92.82) 12 62.82 66.772.20 1.24 2.11 (68.62) 13 90.59 93.79 1.21 0.65 0.77 (95.93) 14 91.4194.88 0.18 0.47 0.65 (97.09) 15 90.91 90.31 0.17 0.56 1.64 (96.18) 1791.45 92.92 0.71 0.56 0.73 (96.81) ¹⁾ As for Example 10 with additionalprotective coating of Aquacoat (Aquacoat Ethylcellulose AquaeousDispersion, 15% w/w, FMC Biopolymer, ECD-30) ²⁾ As for Example 10 withadditional protective coating of Opadry (Opadry AMB dispersion, 20% w/w,Colorcon). ³⁾ As for Example 10 with additional protective coating ofEudragit (Eudragit E PO, Degussa, Roehm Pharma Polymers; SLS, StearicAcid)

Example 56 Linaclotide Tablet Formulation and Stability Testing

Active linaclotide granules were made by fluid bed granulationessentially as described in Example 26 using the reagents described inTable 18. The linaclotide granules were blended with the excipientsdescribed in Table 19 and compressed into tablets with a hardness of ˜4kp.

35 tablets were packaged in a 60 cc bottle with 5 gram desiccant andstored at 40° C./75% RH for up to 3 months or 30° C./65% RH for up to 3months.

Linaclotide content and purity as well as the amount oflinaclotide-related substances were measured essentially as described inExample 21 or by an equivalent method. Results are provided in Table 20.

TABLE 18 Granule, 150 μg Ingredients Function linaclotide/53.7 mggranules Linaclotide API 0.15 mg Mannitol, USP Granule filler 50 mgLeucine, USP Stabilizer 0.64 mg CaCl₂•2H₂O, USP Stabilizer 0.72 mg PVPK30, USP Binder 2.2 mg HCl solution (pH 2.5) — —

TABLE 19 Tablet (200 mg total Ingredients Function weight) Linaclotidegranules Active 53.4 Isomalt, USP Tablet filler 134.1 CroscarmelloseSodium, USP Disintegrant 10 Magnesium stearate, USP Lubricant 1.5 Talc,USP Glidant 1.0

TABLE 20 Condition Time Change in Assay % Total Degradation 40° C./75%RH Initial 100 2.27 40° C./75% RH 1 month 96.2 2.09 40° C./75% RH 2months 102 2.15 40° C./75% RH 3 months 99.5 1.52 30° C./65% RH 3 months100.1 1.19

Example 57 Linaclotide Capsule Formulation

The linaclotide formulation of Example 57 was produced essentially asdescribed in Example 16. Table 21 provides the coating solutioningredients and their theoretical weights (mg/g) and (kg/Batch) for thecomplete Linaclotide Beads Drug Layer Solution. Table 22 provides theingredients and theoretical weights (mg/g) and (kg/Batch) for thepreparation for the Linaclotide Active Beads. The linaclotideformulation was encapsulated in hard gelatin capsules, size 2 (weight 61mg), essentially as described in Example 20. The 150 μg linaclotidecapsules contained 56 mg linaclotide beads (600 μg linaclotide/225 mgbeads) while the 300 μg linaclotide capsules contained 113 mglinaclotide beads (600 μg linaclotide/225 mg beads).

TABLE 21 Theoretical Theoretical Weight Weight Ingredients Function(mg/g) (kg/batch) Linaclotide API 2.67 0.067 CaCl₂•2H₂O, USP, EP, BP, JPStabilizer 15.41 0.385 L-Leucine, USP Stabilizer 6.87 0.172Hydroxypropyl Methylcellulose, Binder 7.00 0.175 USP (Methocel E5Premium LV) Purified Water, USP — — 16.666 HCl (36.5-38.0), NF — — 0.114

TABLE 22 Theoretical Theoretical Weight Weight Ingredients Function(mg/g) (kg/batch) Linaclotide Beads Drug Layer Coating 31.95 0.799Solution solution Microcrystalline cellulose Beads 968.05 24.201 spheresNF (Celphere CP- 305) Final Total: Active beads 1000 25.000 LinaclotideBeads, 600 μg/225 mg)

1. A pharmaceutical composition comprising linaclotide and apharmaceutically acceptable excipient, wherein the chromatographicpurity of the linaclotide decreases by less than 10% after (a) 18 monthsof storage of the pharmaceutical composition at 25° C. at 60% relativehumidity in a sealed container containing a desiccant or (b) 6 months ofstorage of the pharmaceutical composition at 40° C. at 75% relativehumidity in a sealed container containing a desiccant.
 2. Thepharmaceutical composition according to claim 1, wherein thechromatographic purity of the linaclotide decreases by less than 9%, 8%,7%, 6%, 5%, or 4% after (a) 18 months of storage of the pharmaceuticalcomposition at 25° C. at 60% relative humidity in a sealed containercontaining a desiccant or (b) 6 months of storage of the pharmaceuticalcomposition at 40° C. at 75% relative humidity in a sealed containercontaining a desiccant.
 3. A unit dosage form of a pharmaceuticalcomposition comprising linaclotide and a pharmaceutically acceptableexcipient, wherein the chromatographic purity of the linaclotidedecreases by less than 10% after (a) 18 months of storage of the unitdosage form at 25° C. at 60% relative humidity in a sealed containercontaining a desiccant or (b) 6 months of storage of the unit dosageform at 40° C. at 75% relative humidity in a sealed container containinga desiccant.
 4. The unit dosage form according to claim 3, wherein thechromatographic purity of the linaclotide decreases by less than 9%, 8%,7%, 6%, 5% or 4% after (a) 18 months of storage of the unit dosage format 25° C. at 60% relative humidity in a sealed container containing adesiccant or (b) 6 months of storage of the unit dosage form at 40° C.at 75% relative humidity in a sealed container containing a desiccant.5. A sealed container comprising a plurality of unit dosage forms of apharmaceutical composition comprising linaclotide and a pharmaceuticallyacceptable excipient wherein the chromatographic purity of thelinaclotide decreases by less than 10% after (a) 18 months of storage ofthe sealed container containing a desiccant at 25° C. at 60% relativehumidity or (b) 6 months of storage of the sealed container containing adesiccant at 40° C. at 75% relative humidity.
 6. The sealed containeraccording to claim 5, wherein the chromatographic purity of thelinaclotide decreases by less than 9%, 8%, 7%, 6%, 5% or 4% after (a) 18months of storage of the sealed container containing a desiccant at 25°C. at 60% relative humidity or (b) 6 months of storage of the sealedcontainer containing a desiccant at 40° C. at 75% relative humidity. 7.A pharmaceutical composition comprising linaclotide and apharmaceutically acceptable excipient, wherein the assay value forlinaclotide determined on a weight/weight basis decreases by less than10% after (a) 18 months of storage of the pharmaceutical composition at25° C. at 60% relative humidity in a sealed container containing adesiccant or (b) 6 months of storage of the pharmaceutical compositionat 40° C. at 75% relative humidity in a sealed container containing adesiccant.
 8. The pharmaceutical composition according to claim 7,wherein the assay value for the linaclotide decreases by less than 9%,8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% after (a) 18 months of storage of thepharmaceutical composition at 25° C. at 60% relative humidity in asealed container containing a desiccant or (b) 6 months of storage ofthe pharmaceutical composition at 40° C. at 75% relative humidity in asealed container containing a desiccant.
 9. A unit dosage form of apharmaceutical composition comprising linaclotide and a pharmaceuticallyacceptable excipient, wherein the assay value for linaclotide determinedon a weight/weight basis decreases by less than 10% after (a) 18 monthsof storage of the unit dosage form at 25° C. at 60% relative humidity ina sealed container containing a desiccant or (b) 6 months of storage ofthe unit dosage form at 40° C. at 75% relative humidity in a sealedcontainer containing a desiccant.
 10. The unit dosage form according toclaim 9, wherein the assay value for the linaclotide decreases by lessthan 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% after (a) 18 months of storageof the unit dosage form at 25° C. at 60% relative humidity in a sealedcontainer containing a desiccant or (b) 6 months of storage of the unitdosage form at 40° C. at 75% relative humidity in a sealed containercontaining a desiccant.
 11. A sealed container comprising a plurality ofunit dosage forms of a pharmaceutical composition comprising linaclotideand a pharmaceutically acceptable excipient wherein the assay value forlinaclotide in the unit dosage forms determined on a weight/weight basisdecreases by less than 10% after (a) 18 months of storage of the sealedcontainer containing a desiccant at 25° C. at 60% relative humidity or(b) 6 months of storage the sealed container containing a desiccant at40° C. at 75% relative humidity.
 12. The sealed container according toclaim 11, wherein the assay value for the linaclotide decreases by lessthan 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% after (a) 18 months of storagethe sealed container containing a desiccant at 25° C. at 60% relativehumidity or (b) 6 months of storage the sealed container containing adesiccant at 40° C. at 75% relative humidity.
 13. The unit dosage formaccording to any one of claim 3-4 or 9-10, wherein each unit dosage formcontains from 50 μg to 2 mg linaclotide.
 14. The sealed containeraccording to any one of claim 5-6 or 11-12, wherein each unit dosageform contains from 50 μg to 2 mg linaclotide.
 15. The pharmaceuticalcomposition according to either of claim 1 or 2, wherein thechromatographic purity of the linaclotide decreases by less than 10%after (a) 24 months of storage of the pharmaceutical composition at 25°C. at 60% relative humidity in a sealed container containing a desiccantor (b) 6 months of storage of the pharmaceutical composition at 40° C.at 75% relative humidity in a sealed container containing a desiccant.16. The unit dosage form according to either of claim 3 or 4, whereinthe chromatographic purity of the linaclotide decreases by less than 10%after (a) 24 months of storage of the unit dosage form at 25° C. at 60%relative humidity in a sealed container containing a desiccant or (b) 6months of storage of the unit dosage form at 40° C. at 75% relativehumidity in a sealed container containing a desiccant.
 17. The sealedcontainer according to either of claim 5 or 6, wherein thechromatographic purity of the linaclotide decreases by less than 10%after (a) 24 months of storage of the pharmaceutical composition at 25°C. at 60% relative humidity in a sealed container containing a desiccantor (b) 6 months of storage of the pharmaceutical composition at 40° C.at 75% relative humidity in a sealed container containing a desiccant.18. The pharmaceutical composition according to either of claim 7 or 8,wherein the assay value of the linaclotide decreases by less than 10%after (a) 24 months of storage of the pharmaceutical composition at 25°C. at 60% relative humidity in a sealed container containing a desiccantor (b) 6 months of storage of the pharmaceutical composition at 40° C.at 75% relative humidity in a sealed container containing a desiccant.19. The unit dosage form according to either of claim 9 or 10, whereinthe assay value of the linaclotide decreases by less than 10% after (a)24 months of storage of the unit dosage form at 25° C. at 60% relativehumidity in a sealed container containing a desiccant or (b) 6 months ofstorage of the unit dosage form at 40° C. at 75% relative humidity in asealed container containing a desiccant.
 20. The sealed containeraccording to either of claim 11 or 12, wherein the assay value of thelinaclotide decreases by less than 10% after (a) a first 24 months ofstorage of the sealed container containing a desiccant at 25° C. at 60%relative humidity or (b) 6 months of storage of the sealed containercontaining a desiccant at 40° C. at 75% relative humidity.
 21. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier, linaclotide and one or more agents selected from (i) a cationselected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺, or (ii) asterically hindered primary amine, wherein the agent improves at leastone attribute of the composition, relative to a pharmaceuticalcomposition without said agent, after (a) a first 18 months of storageof the pharmaceutical composition at 25° C. at 60% relative humidity ina sealed container containing a desiccant or (b) a first 6 months ofstorage of the pharmaceutical composition at 40° C. at 75% relativehumidity in a sealed container containing a desiccant, wherein saidattribute is selected from a decrease in the rate of degradation oflinaclotide as measured by linaclotide content, a decrease in the rateof degradation of linaclotide as measured by chromatographic purity oflinaclotide, a decrease in the amount of a linaclotide oxidation productrelative to the amount of linaclotide, a decrease in the amount of alinaclotide hydrolysis product relative to the amount of linaclotide, ora decrease in the amount of a linaclotide formaldehyde imine product oflinaclotide relative to the amount of linaclotide.
 22. Thepharmaceutical composition according to claim 21, wherein said agent isMg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺.
 23. The pharmaceuticalcomposition according to claim 22, wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺,K⁺, Na⁺ or Al³⁺ is provided as magnesium acetate, magnesium chloride,magnesium phosphate, magnesium sulfate, calcium acetate, calciumchloride, calcium phosphate, calcium sulfate, zinc acetate, zincchloride, zinc phosphate, zinc sulfate, manganese acetate, manganesechloride, manganese phosphate, manganese sulfate, potassium acetate,potassium chloride, potassium phosphate, potassium sulfate, sodiumacetate, sodium chloride, sodium phosphate, sodium sulfate, aluminumacetate, aluminum chloride, aluminum phosphate or aluminum sulfate. 24.The pharmaceutical composition according to claim 23, wherein said Mg²⁺,Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ is provided as magnesium chloride,calcium chloride, calcium phosphate, calcium sulfate, zinc acetate,manganese chloride, potassium chloride, sodium chloride or aluminumchloride.
 25. The pharmaceutical composition according to claim 22,wherein said agent is Mg²⁺, Ca²⁺ or Zn²⁺.
 26. The pharmaceuticalcomposition according to claim 25, wherein said Mg²⁺, Ca²⁺ or Zn²⁺ isprovided as magnesium chloride, calcium chloride or zinc acetate. 27.The pharmaceutical composition according to claim 22, wherein said agentis Ca²⁺.
 28. The pharmaceutical composition according to claim 27,wherein said Ca²⁺ is provided as calcium chloride.
 29. Thepharmaceutical composition according to claim 21, wherein said agent isa sterically hindered primary amine.
 30. The pharmaceutical compositionaccording to claim 29, wherein the sterically hindered primary amine isan amino acid.
 31. The pharmaceutical composition according to claim 30,wherein the amino acid is a naturally-occurring amino acid.
 32. Thepharmaceutical composition according to claim 31, wherein thenaturally-occurring amino acid is histidine, phenylalanine, alanine,glutamic acid, aspartic acid, glutamine, leucine, methionine,asparagine, tyrosine, threonine, isoleucine, tryptophan or valine. 33.The pharmaceutical composition according to claim 32, wherein thenaturally-occurring amino acid is histidine, phenylalanine, leucine,methionine, isoleucine, tryptophan or valine.
 34. The pharmaceuticalcomposition according to claim 33, wherein the naturally-occurring aminoacid is leucine, isoleucine, alanine or methionine.
 35. Thepharmaceutical composition according to claim 34, wherein thenaturally-occurring amino acid is leucine or methionine.
 36. Thepharmaceutical composition according to claim 35, wherein thenaturally-occurring amino acid is leucine.
 37. The pharmaceuticalcomposition according to claim 30, wherein the sterically hinderedprimary amine is a non-naturally occurring amino acid or an amino acidderivative.
 38. The pharmaceutical composition according to claim 37,wherein the non-naturally occurring amino acid is 1-aminocyclohexanecarboxylic acid, lanthanine or theanine.
 39. The pharmaceuticalcomposition according to claim 29, wherein the sterically hinderedprimary amine has the formula:

wherein R₁, R₂ and R₃ are independently selected from: H; —C(O)OH; C₁-C₆alkyl, optionally substituted by —CO₂H, —CONH₂, or a 5-10 membered arylor heteroaryl; C₁-C₆ alkoxyalkyl; or C₁-C₆ thioalkoxyalkyl, wherein anyof the alkyl or aryl groups above can be singly or multiply substitutedwith halogen or —NH₂, and provided that no more than two of R₁, R₂ andR₃ are H.
 40. The pharmaceutical composition according to claim 39,wherein the sterically hindered primary amine is cyclohexylamine or2-methylbutylamine.
 41. The pharmaceutical composition according toclaim 29, wherein the sterically hindered primary amine is a polymericamine.
 42. The pharmaceutical composition according to claim 41, whereinthe polymeric amine is chitosan.
 43. The pharmaceutical compositionaccording to any one of claims 29-42, wherein said pharmaceuticalcomposition further comprises Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺.44. The pharmaceutical composition according to claim 43, wherein saidMg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ is provided as magnesiumacetate, magnesium chloride, magnesium phosphate, magnesium sulfate,calcium acetate, calcium chloride, calcium phosphate, calcium sulfate,zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganeseacetate, manganese chloride, manganese phosphate, manganese sulfate,potassium acetate, potassium chloride, potassium phosphate, potassiumsulfate, sodium acetate, sodium chloride, sodium phosphate, sodiumsulfate, aluminum acetate, aluminum chloride, aluminum phosphate oraluminum sulfate.
 45. The pharmaceutical composition according to claim44, wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na or Al³⁺ is provided asmagnesium chloride, calcium chloride, calcium phosphate, calciumsulfate, zinc acetate, manganese chloride, potassium chloride, sodiumchloride or aluminum chloride.
 46. The pharmaceutical compositionaccording to any one of claims 29-42, wherein said pharmaceuticalcomposition further comprises Mg²⁺, Ca²⁺ or Zn²⁺.
 47. The pharmaceuticalcomposition according to claim 46, wherein said Mg²⁺, Ca²⁺ or Zn²⁺ isprovided as magnesium chloride, calcium chloride or zinc acetate. 48.The pharmaceutical composition according to claim 46, wherein saidpharmaceutical composition further comprises Ca²⁺.
 49. Thepharmaceutical composition according to claim 48, wherein said Ca²⁺ isprovided as calcium chloride.
 50. The pharmaceutical compositionaccording to any one of claims 21-49, further comprising an antioxidant.51. The pharmaceutical composition according to claim 50, wherein saidantioxidant is BHA, vitamin E or propyl gallate.
 52. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier,linaclotide, a cation selected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ orAl³⁺, and a sterically hindered primary amine.
 53. The pharmaceuticalcomposition according to claim 52, wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺,K⁺, Na⁺ or Al³⁺ is provided as magnesium acetate, magnesium chloride,magnesium phosphate, magnesium sulfate, calcium acetate, calciumchloride, calcium phosphate, calcium sulfate, zinc acetate, zincchloride, zinc phosphate, zinc sulfate, manganese acetate, manganesechloride, manganese phosphate, manganese sulfate, potassium acetate,potassium chloride, potassium phosphate, potassium sulfate, sodiumacetate, sodium chloride, sodium phosphate, sodium sulfate, aluminumacetate, aluminum chloride, aluminum phosphate or aluminum sulfate. 54.The pharmaceutical composition according to claim 53, wherein said Mg²⁺,Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ is provided as magnesium chloride,calcium chloride, calcium phosphate, calcium sulfate, zinc acetate,manganese chloride, potassium chloride, sodium chloride or aluminumchloride.
 55. The pharmaceutical composition according to claim 52,wherein said cation is selected from Mg²⁺, Ca²⁺ or Zn²⁺.
 56. Thepharmaceutical composition according to claim 55, wherein said Mg²⁺,Ca²⁺ or Zn²⁺ is provided as magnesium chloride, calcium chloride or zincacetate.
 57. The pharmaceutical composition according to claim 55,wherein said cation is Ca²⁺.
 58. The pharmaceutical compositionaccording to claim 57, wherein said Ca²⁺ is provided as calciumchloride.
 59. The pharmaceutical composition according to any one ofclaims 52-56, wherein the sterically hindered primary amine is an aminoacid.
 60. The pharmaceutical composition according to claim 59, whereinthe amino acid is a naturally-occurring amino acid.
 61. Thepharmaceutical composition according to claim 60, wherein thenaturally-occurring amino acid is histidine, phenylalanine, alanine,glutamic acid, aspartic acid, glutamine, leucine, methionine,asparagine, tyrosine, threonine, isoleucine, tryptophan, methionine orvaline.
 62. The pharmaceutical composition according to claim 61,wherein the naturally-occurring amino acid is histidine, phenylalanine,leucine, methionine, isoleucine, tryptophan or valine.
 63. Thepharmaceutical composition according to claim 62, wherein thenaturally-occurring amino acid is leucine, isoleucine, alanine ormethionine.
 64. The pharmaceutical composition of claim 63, wherein thenaturally-occurring amino acid is leucine.
 65. The pharmaceuticalcomposition according to claim 52, wherein the sterically hinderedprimary amine is a non-naturally occurring amino acid or an amino acidderivative.
 66. The pharmaceutical composition according to claim 65,wherein the non-naturally occurring amino acid is 1-aminocyclohexanecarboxylic acid, lanthanine or theanine.
 67. The pharmaceuticalcomposition according to claim 52, wherein the sterically hinderedprimary amine has the formula:

wherein R₁, R₂ and R₃ are independently selected from: H; —C(O)OH; C₁-C₆alkyl, optionally substituted by —CO₂H, —CONH₂, or a 5-10 membered arylor heteroaryl; C₁-C₆ alkoxyalkyl; or C₁-C₆ thioalkoxyalkyl, wherein anyof the alkyl or aryl groups above can be singly or multiply substitutedwith halogen or —NH₂, and provided that no more than two of R₁, R₂ andR₃ are H.
 68. The pharmaceutical composition according to claim 67,wherein the sterically hindered primary amine is cyclohexylamine or2-methylbutylamine.
 69. The pharmaceutical composition according toclaim 52, wherein the sterically hindered primary amine is a polymericamine.
 70. The pharmaceutical composition according to claim 69, whereinthe sterically hindered primary amine is chitosan.
 71. Thepharmaceutical composition according to any one of claims 52-70, furthercomprising a pharmaceutically acceptable glidant, lubricant or additivethat acts as both a glidant and lubricant.
 72. The pharmaceuticalcomposition according to any one of claims 52-71 further comprising anantioxidant.
 73. The pharmaceutical composition according to claim 72,wherein said antioxidant is BHA, vitamin E or propyl gallate.
 74. Thepharmaceutical composition according to any one of claims 52-73, furthercomprising a pharmaceutically acceptable binder.
 75. The pharmaceuticalcomposition according to claim 74 wherein the pharmaceuticallyacceptable binder is selected from polyvinyl alcohol,polyvinylpyrrolidone (povidone), a starch, maltodextrin or a celluloseether.
 76. The pharmaceutical composition of claim 75 wherein thepharmaceutically acceptable binder is a cellulose ether.
 77. Thepharmaceutical composition of claim 76 wherein the cellulose ether isselected from: methylcellulose, ethylcellulose, carboxymethylcellulose,hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropylcellulose and hydroxypropyl methylcellulose.
 78. The pharmaceuticalcomposition of any of claims 52-77, further comprising apharmaceutically acceptable filler.
 79. The pharmaceutical compositionaccording to claim 78, wherein the pharmaceutically acceptable filler iscellulose, isomalt, mannitol or dibasic calcium phosphate.
 80. Thepharmaceutical composition of claim 79 wherein the cellulose is selectedfrom microfine cellulose and microcrystalline cellulose.
 81. Thepharmaceutical composition of any of claims 78-80, wherein thepharmaceutically acceptable filler comprises particles having an averagediameter between 150 μm and 1000 μm.
 82. The pharmaceutical compositionof any of claims 52-81, wherein pharmaceutical composition comprises afiller and the weight ratio of linaclotide to pharmaceuticallyacceptable filler is between 1:25 and 1:2,500.
 83. The pharmaceuticalcomposition according to claim 82, wherein the weight ratio oflinaclotide to pharmaceutically acceptable filler is between 1:100 and1:2000.
 84. The pharmaceutical composition according to claim 83,wherein the weight ratio of linaclotide to pharmaceutically acceptablefiller is between 1:100 and 1:1000.
 85. The pharmaceutical compositionof any of claims 52-84 wherein the sterically hindered amine is leucineand the molar ratio of leucine to linaclotide is at least 10:1.
 86. Thepharmaceutical composition of claim 85 wherein the molar ratio ofleucine to linaclotide is at least 20:1.
 87. The pharmaceuticalcomposition of claim 86 wherein the molar ratio of leucine tolinaclotide is at least 30:1.
 88. The pharmaceutical composition of anyof claims 52-87 wherein the cation is Ca²⁺ and the molar ratio of Ca²⁺to leucine is at least 1:1.
 89. The pharmaceutical composition of claim88, wherein the molar ratio of Ca²⁺ to leucine is at least 1.5:1. 90.The pharmaceutical composition of claim 89 wherein the molar ratio ofCa²⁺ to leucine is at least 2:1.
 91. The pharmaceutical compositionaccording to any one of claims 52-84, wherein the molar ratio ofcation:sterically hindered primary amine:linaclotide is 40-100:20-50:1.92. The pharmaceutical composition according to claim 91, wherein thecation is Ca²⁺ and the sterically hindered primary amine is leucine. 93.The pharmaceutical composition according to claim 92, wherein the molarratio of Ca²⁺:leucine:linaclotide is 100:30:1, 80:40:1, 80:30:1,80:20:1, 60:30:1, 60:20:1, 50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1,10:5:1, 5:10:1 or 5:5:1.
 94. The pharmaceutical composition according toclaim 93, wherein the molar ratio of Ca²⁺:leucine:linaclotide is60:30:1.
 95. The pharmaceutical composition according to any one ofclaims 92-94, wherein Ca²⁺ is provided as CaCl₂.
 96. A unit dosage formcomprising the pharmaceutical composition according to any one of claims92-95.
 97. The unit dosage form according to claim 96, wherein each unitdosage form comprises 50 μg to 1 mg linaclotide.
 98. The unit dosageform according to claim 97, wherein each unit dosage form is a capsuleor tablet, and wherein said unit dosage form comprises 67.5 μg, 100 μg,133 μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg or 600 μglinaclotide.
 99. A method for preparing a pharmaceutical compositioncomprising linaclotide or a salt thereof, the method comprising: (a)providing an aqueous solution comprising: (i) linaclotide or apharmaceutically acceptable salt thereof (ii) one or more of a cationselected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ or a stericallyhindered primary amine; and, (iii) optionally, a pharmaceuticallyacceptable binder; and (b) applying the aqueous solution to apharmaceutically acceptable filler to generate linaclotide-coatedfiller.
 100. The method of claim 99, wherein the aqueous solutioncomprises a sterically hindered primary amine.
 101. The method of claim99, wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ is provided asmagnesium acetate, magnesium chloride, magnesium phosphate, magnesiumsulfate, calcium acetate, calcium chloride, calcium phosphate, calciumsulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate,manganese acetate, manganese chloride, manganese phosphate, manganesesulfate, potassium acetate, potassium chloride, potassium phosphate,potassium sulfate, sodium acetate, sodium chloride, sodium phosphate,sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphateor aluminum sulfate.
 102. The method of claim 101, wherein said Mg²⁺,Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ is provided as magnesium chloride,calcium chloride, calcium phosphate, calcium sulfate, zinc acetate,manganese chloride, potassium chloride, sodium chloride or aluminumchloride.
 103. The method of claim 99, wherein said cation is selectedfrom Mg²⁺, Ca²⁺ or Zn²⁺.
 104. The method of claim 103, wherein saidMg²⁺, Ca²⁺ or Zn²⁺ is provided as magnesium chloride, calcium chlorideor zinc acetate.
 105. The method of claim 103, wherein said cation isCa²⁺.
 106. The method of claim 105, wherein said Ca²⁺ is provided ascalcium chloride.
 107. The method of claim 99, wherein the stericallyhindered primary amine is an amino acid.
 108. The method of claim 107,wherein the amino acid is a naturally-occurring amino acid.
 109. Themethod of claim 108, wherein the naturally-occurring amino acid ishistidine, phenylalanine, alanine, glutamic acid, aspartic acid,glutamine, leucine, methionine, asparagine, tyrosine, threonine,isoleucine, tryptophan, methionine or valine.
 110. The method of claim109, wherein the naturally-occurring amino acid is histidine,phenylalanine, leucine, methionine, isoleucine, tryptophan or valine.111. The method of claim 110, wherein the naturally-occurring amino acidis leucine, isoleucine, alanine or methionine.
 112. The method of claim111, wherein the naturally-occurring amino acid is leucine.
 113. Themethod of claim 99, wherein the sterically hindered primary amine is anon-naturally occurring amino acid or an amino acid derivative.
 114. Themethod of claim 113, wherein the non-naturally occurring amino acid is1-aminocyclohexane carboxylic acid, lanthanine or theanine.
 115. Themethod of claim 99, wherein the sterically hindered primary amine hasthe formula:

wherein R_(I), R₂ and R₃ are independently selected from: H; —C(O)OH;C₁-C₆ alkyl, optionally substituted by —CO₂H, —CONH₂, or a 5-10 memberedaryl or heteroaryl; C₁-C₆ alkoxyalkyl; or C₁-C₆ thioalkoxyalkyl, whereinany of the alkyl or aryl groups above can be singly or multiplysubstituted with halogen or —NH₂, and provided that no more than two ofR₁, R₂ and R₃ are H.
 116. The method of claim 115, wherein thesterically hindered primary amine is cyclohexylamine or2-methylbutylamine.
 117. The method of claim 99, wherein the stericallyhindered primary amine is a polymeric amine.
 118. The method of claim117, wherein the sterically hindered primary amine is chitosan.
 119. Themethod according to claims 99-118, wherein the aqueous solution furthercomprises an antioxidant.
 120. The method of claim 119, wherein saidantioxidant is BHA, BHT, vitamin E, propyl gallate, ascorbic acid andsalts or esters thereof, tocopherol and esters thereof, alpha-lipoicacid or beta-carotene.
 121. The method of claim 99, wherein the aqueoussolution comprises both the cation selected from Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺,K⁺, Na⁺ or Al³⁺ and the sterically hindered primary amine.
 122. Themethod of claim 121, wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ orAl³⁺ is provided as magnesium acetate, magnesium chloride, magnesiumphosphate, magnesium sulfate, calcium acetate, calcium chloride, calciumphosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate,zinc sulfate, manganese acetate, manganese chloride, manganesephosphate, manganese sulfate, potassium acetate, potassium chloride,potassium phosphate, potassium sulfate, sodium acetate, sodium chloride,sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride,aluminum phosphate or aluminum sulfate.
 123. The method of claim 122,wherein said Mg²⁺, Ca²⁺, Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ is provided asmagnesium chloride, calcium chloride, calcium phosphate, calciumsulfate, zinc acetate, manganese chloride, potassium chloride, sodiumchloride or aluminum chloride.
 124. The method of claim 121, whereinsaid cation is selected from Mg²⁺, Ca²⁺ or Zn²⁺.
 125. The method ofclaim 124, wherein said Mg²⁺, Ca²⁺ or Zn²⁺ is provided as magnesiumchloride, calcium chloride or zinc acetate.
 126. The method of claim124, wherein said cation is Ca²⁺.
 127. The method of claim 126, whereinsaid Ca²⁺ is provided as calcium chloride.
 128. The method of claim 121,wherein the sterically hindered primary amine is an amino acid.
 129. Themethod of claim 128, wherein the amino acid is a naturally-occurringamino acid.
 130. The method of claim 129, wherein thenaturally-occurring amino acid is histidine, phenylalanine, alanine,glutamic acid, aspartic acid, glutamine, leucine, methionine,asparagine, tyrosine, threonine, isoleucine, tryptophan, methionine orvaline.
 131. The method of claim 130, wherein the naturally-occurringamino acid is histidine, phenylalanine, leucine, methionine, isoleucine,tryptophan or valine.
 132. The method of claim 131, wherein thenaturally-occurring amino acid is leucine, isoleucine, alanine ormethionine.
 133. The method of claim 132, wherein thenaturally-occurring amino acid is leucine.
 134. The method of claim 121,wherein the sterically hindered primary amine is a non-naturallyoccurring amino acid or an amino acid derivative.
 135. The method ofclaim 134, wherein the non-naturally occurring amino acid is1-aminocyclohexane carboxylic acid, lanthanine or theanine.
 136. Themethod of claim 121, wherein the sterically hindered primary amine hasthe formula:

wherein R₁, R₂ and R₃ are independently selected from: H; —C(O)OH; C₁-C₆alkyl, optionally substituted by —CO₂H, —CONH₂, or a 5-10 membered arylor heteroaryl; C₁-C₆ alkoxyalkyl; or C₁-C₆ thioalkoxyalkyl, wherein anyof the alkyl or aryl groups above can be singly or multiply substitutedwith halogen or —NH₂, and provided that no more than two of R₁, R₂ andR₃ are H.
 137. The method of claim 136, wherein the sterically hinderedprimary amine is cyclohexylamine or 2-methylbutylamine.
 138. The methodof claim 121, wherein the sterically hindered primary amine is apolymeric amine.
 139. The method of claim 138, wherein the stericallyhindered primary amine is chitosan.
 140. The method according to claims121-140, wherein the aqueous solution further comprises an antioxidant.141. The method of claim 140, wherein said antioxidant is BHA, BHT,vitamin E, propyl gallate, ascorbic acid and salts or esters thereof,tocopherol and esters thereof, alpha-lipoic acid or beta-carotene. 142.The method of claim 141 wherein the antioxidant is BHA.
 143. The methodof any one of claims 99-142, wherein the binder is present and isselected from polyvinyl alcohol, polyvinylpyrrolidone (povidone), astarch, maltodextrin or a cellulose ether.
 144. The method of claim 143wherein the pharmaceutically acceptable binder is a cellulose ether.145. The method of claim 144 wherein the cellulose ether is selectedfrom: methylcellulose, ethylcellulose, carboxymethylcellulose,hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropylcellulose and hydroxypropyl methylcellulose.
 146. The method of any oneof claims 99-145, wherein the filler is selected from cellulose,isomalt, mannitol or dibasic calcium phosphate.
 147. The method of claim146, wherein the filler is microfine cellulose or microcrystallinecellulose.
 148. The method of any one of claims 99-147, wherein theaqueous solution is applied to the filler by spraying.
 149. The methodof any one of claims 99-147, wherein the aqueous solution is applied tothe filler by mixing.
 150. The method of any one of claims 99-149,wherein the weight ratio of linaclotide to pharmaceutically acceptablefiller is between 1:100 and 1:2500.
 151. The method according to claim150, wherein the weight ratio of linaclotide to pharmaceuticallyacceptable filler is between 1:100 and 1:1000.
 152. The method accordingto any one of claims 121-151, wherein the molar ratio ofcation:sterically hindered primary amine:linaclotide is 40-100:20-30:1.153. The method according to claim 152, wherein the cation is Ca²⁺. 154.The method according to claim 152, wherein the sterically hinderedprimary amine is leucine.
 155. The method according to claim 152,wherein the cation is Ca²⁺ and the sterically hindered primary amine isleucine, and the molar ratio of Ca²⁺:leucine:linaclotide is 100:30:1,80:40:1, 80:30:1, 80:20:1, 60:30:1, 60:20:1, 50:30:1, 50:20:1, 40:20:1,20:20:1, 10:10:1, 10:5:1 or 5:5:1.
 156. The method according to claim155, wherein the molar ratio of Ca²⁺:leucine:linaclotide is 60:30:1.157. The method according to any one of claims 152-156, wherein thepharmaceutically acceptable filler is selected from cellulose, isomalt,mannitol or dibasic calcium phosphate.
 158. The method according toclaim 157, wherein the pharmaceutically acceptable filler is microfinecellulose or microcrystalline cellulose.
 159. The method according toany one of claims 152-158, wherein the pharmaceutically acceptablebinder is polyvinyl alcohol, polyvinylpyrrolidone (povidone), a starch,maltodextrin or a cellulose ether.
 160. The method of claim 159 whereinthe pharmaceutically acceptable binder is a cellulose ether.
 161. Themethod according to claim 160, wherein the pharmaceutically acceptablebinder is methylcellulose, ethylcellulose, carboxymethylcellulose,hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropylcellulose and hydroxypropyl methylcellulose.
 162. The method accordingto any one of claims 99-161, wherein the method further comprises (c)applying a coating to the linaclotide-coated filler.
 163. The methodaccording to claim 162, wherein said coating is selected from AQUACOAT,EUDRAGIT or OPADRY.
 164. The method according to any one of claims99-163, wherein the linaclotide-coated filler is mixed with one or morepharmaceutically acceptable additives.
 165. The method according to anyone of claims 99-164, further comprising tableting or encapsulating thelinaclotide-coated filler in a tablet or capsule, respectively.
 166. Themethod according to claim 165, wherein said linaclotide-coated filler isencapsulated in a capsule.
 167. The method according to claim 166,wherein the capsule is a gelatin capsule.
 168. The method according toany one of claims 165-167, wherein each capsule or tablet contains 50 μgto 1 mg linaclotide.
 169. The method according to claim 168, whereineach capsule or tablet contains 50 μg, 67.5 μg, 100 μg, 133 μg, 150 μg,200 μg, 266 μg, 300 μg, 400 μg, 500 μg or 600 μg linaclotide.
 170. Themethod according to claim 169, wherein each capsule or tablet contains67.5 μg, 133 μg, 150 μg, 266 μg or 300 μg linaclotide.
 171. A method fortreating a patient suffering from impaired intestinal motility,irritable bowel syndrome, constipation, pain associated withconstipation, dyspepsia, gastroparesis, chronic intestinal pseudoobstruction, Crohn's disease, ulcerative colitis, or inflammatory boweldisease, comprising administering to the patient an effective amount ofthe pharmaceutical composition according to any one of claims 1-98. 172.The method according to claim 171, wherein said irritable bowel syndromeis constipation-predominant irritable bowel syndrome or alternatingirritable bowel syndrome.
 173. The method according to claim 172,wherein said irritable bowel syndrome is constipation-predominantirritable bowel syndrome.
 174. The method according to claim 171,wherein said constipation is chronic constipation, idiopathicconstipation, post-operative ileus, or constipation caused by opiateuse.
 175. The method according to claim 174, wherein said constipationis chronic constipation.
 176. The method according to any one of claims171-175, wherein the pharmaceutical composition contains 50 μg to 1 mglinaclotide.
 177. The method according to claim 176, wherein thepharmaceutical composition contains 50 μg, 67.5 μg, 100 μg, 133 μg, 150μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg or 600 μg linaclotide. 178.The method according to claim 177, wherein the pharmaceuticalcomposition contains 67.5 μg, 133 μg, 150 μg, 266 μg or 300 μglinaclotide.
 179. The method according to any one of claims 171-178,wherein the pharmaceutical composition is administered once daily, twicedaily, three times daily or four times daily.
 180. The method accordingto claim 179, wherein the pharmaceutical composition is administeredonce daily or twice daily.
 181. The method according to claim 180,wherein the pharmaceutical composition is administered once daily. 182.The method according to claim 181, wherein the pharmaceuticalcomposition is administered once daily as one or two tablets orcapsules.
 183. The method according to claim 182, wherein thepharmaceutical composition is administered as a single tablet orcapsule.
 184. A composition that is at least 10%, at least 20%, at least30%, at least 40% or at least 50% by weight a hydrolysis product oflinaclotide.
 185. The composition of claim 184 wherein the compositionis at least 60%, at least 70% or at least 80% by weight a hydrolysisproduct of linaclotide.
 186. The composition of claim 185 wherein thecomposition is at least 90%, at least 95% or at least 98% by weight ahydrolysis product of linaclotide.
 187. A composition that is at least10%, at least 20%, at least 30%, at least 40% or at least 50% by weightan oxidation product of linaclotide.
 188. The composition of claim 187wherein the composition at least 60%, at least 70% or at least 80% byweight an oxidation product of linaclotide.
 189. The composition ofclaim 188 wherein the composition is at least 90%, at least 95% or atleast 98% by weight an oxidation product of linaclotide.
 190. Acomposition that is at least 10%, at least 20%, at least 30%, at least40% or at least 50% by weight a formaldehyde imine product oflinaclotide.
 191. The composition of claim 190 wherein the compositionis at least 60%, at least 70% or at least 80% by weight a formaldehydeimine product of linaclotide.
 192. The composition of claim 191 whereinthe composition is at least 90%, at least 95% or at least 98% by weighta formaldehyde imine product of linaclotide.
 193. A method for assessingthe purity of a pharmaceutical formulation comprising linaclotide, themethod comprising: (a) providing a sample of the pharmaceuticalformulation comprising linaclotide; (b) measuring the amount of at leastone degradation product in the sample selected from the group consistingof an oxidation product of linaclotide, a hydrolysis product oflinaclotide and a formaldehyde imine product of linaclotide; and (c)comparing the measured amount of the oxidation product of linaclotide,the hydrolysis product of linaclotide and/or the formaldehyde imineproduct of linaclotide to a reference standard amount of the oxidationproduct of linaclotide, the hydrolysis product of linaclotide or theformaldehyde imine product to assess the purity of the pharmaceuticalformulation.
 194. A method for identifying an impurity in a samplecomprising linaclotide comprising: (a) providing a reference samplecomprising a reference marker and linaclotide; (b) carrying out HPLC onthe reference sample to determine the relative retention time of thereference marker compared to linaclotide; (c) carrying out HPLC on thesample comprising linaclotide to determine the relative retention timeof the impurity compared to linaclotide; (d) comparing the relativeretention times determined in steps (b) and (c); where, if the relativeretention times are substantially the same, the impurity is identifiedas being the same as the reference marker; wherein the reference markeris selected from the group consisting of an oxidation product oflinaclotide; a hydrolysis product of linaclotide or a formaldehyde imineproduct of linaclotide.
 195. A method for determining the amount of animpurity in a sample of linaclotide comprising: (a) adding a knownamount of a reference sample to the linaclotide sample; (b) subjectingthe linaclotide sample to HPLC; (c) identifying and measuring the areaof an HPLC peak associated with the impurity; (d) identifying andmeasuring the area of an HPLC peak associated with the referencestandard; (e) calculating the amount of the impurity in the linaclotidesample based on the results of step (c) and (d); wherein the referencesample is selected from the group consisting of an oxidation product oflinaclotide; a hydrolysis product of linaclotide or a formaldehyde imineproduct of linaclotide.
 196. A method for determining the amount of animpurity in a sample of linaclotide comprising: (a) providing a sampleof linaclotide containing an unknown concentration of the impurity; (b)providing a sample of a known concentration of the impurity; (c)subjecting at least a portion of the linaclotide sample and at least aportion of the impurity sample to HPLC; (d) measuring the area of theimpurity peaks obtained from the linaclotide sample and the impuritysample; and (e) calculating the amount of the impurity in thelinaclotide sample based on the measurements of step (d); wherein theimpurity is selected from the group consisting of an oxidation productof linaclotide; a hydrolysis product of linaclotide or a formaldehydeimine product of linaclotide.
 197. The pharmaceutical compositionaccording to any one of claim 1, 2, 15 or 21-95, the unit dosage formaccording to any one of claim 3, 4, 16 or 96-98, or the sealed containeraccording to any one of claim 5, 6, 17 or 238, wherein a hydrolysisproduct having a structure of

comprises less than 2% by weight compared to the weight of thelinaclotide.
 198. The pharmaceutical composition, unit dosage form orsealed container according to claim 197, wherein the hydrolysis productcomprises less than 1% by weight or less than 0.5% by weight compared tothe weight of the linaclotide.
 199. The pharmaceutical composition, unitdosage form or sealed container according to claim 198, wherein thehydrolysis product comprises less than 0.1% by weight or less than 0.05%by weight compared to the weight of the linaclotide.
 200. Thepharmaceutical composition according to any one of claim 1, 2, 15 or21-95, the unit dosage form according to any one of claim 3, 4, 16 or96-98, or the sealed container according to any one of claim 5, 6, 17 or238, wherein a formaldehyde imine product having a structure of

comprises less than 2% by weight compared to the weight of thelinaclotide.
 201. The pharmaceutical composition, unit dosage form orsealed container according to claim 200, wherein the formaldehyde imineproduct comprises less than 1% by weight or less than 0.5% by weightcompared to the weight of the linaclotide.
 202. The pharmaceuticalcomposition, unit dosage form or sealed container according to claim201, wherein the formaldehyde imine product comprises less than 0.1% byweight or less than 0.05% by weight compared to the weight of thelinaclotide.
 203. The pharmaceutical composition according to any one ofclaim 1, 2, 15 or 21-95, the unit dosage form according to any one ofclaim 3, 4, 16 or 96-98, or the sealed container according to any one ofclaim 5, 6, 17 or 238, wherein a linaclotide oxidation product having amolecular weight of 1542.8 comprises less than 2% by weight compared tothe weight of the linaclotide.
 204. The pharmaceutical composition, unitdosage form or sealed container according to claim 203, wherein thelinaclotide oxidation product comprises less than 1% by weight or lessthan 0.5% by weight compared to the weight of the linaclotide.
 205. Thepharmaceutical composition, unit dosage form or sealed containeraccording to claim 204, wherein the linaclotide oxidation productcomprises less than 0.1% by weight or less than 0.05% by weight comparedto the weight of the linaclotide.
 206. A pharmaceutical compositioncomprising linaclotide and a pharmaceutically acceptable excipient,wherein the chromatographic purity of the linaclotide is greater than orequal to 90% after (a) 18 months of storage of the pharmaceuticalcomposition at 25° C. at 60% relative humidity in a sealed containercontaining a desiccant or (b) 6 months of storage of the pharmaceuticalcomposition at 40° C. at 75% relative humidity in a sealed containercontaining a desiccant.
 207. The pharmaceutical composition according toclaim 206, wherein the chromatographic purity of the linaclotide isgreater than or equal to 91%, 92%, 93%, 94%, 95% or 96% after (a) 18months of storage of the pharmaceutical composition at 25° C. at 60%relative humidity in a sealed container containing a desiccant or (b) 6months of storage of the pharmaceutical composition at 40° C. at 75%relative humidity in a sealed container containing a desiccant.
 208. Aunit dosage form of a pharmaceutical composition comprising linaclotideand a pharmaceutically acceptable excipient, wherein the chromatographicpurity of the linaclotide is greater than or equal to 90% after (a) 18months of storage of the unit dosage form at 25° C. at 60% relativehumidity in a sealed container containing a desiccant or (b) 6 months ofstorage of the unit dosage form at 40° C. at 75% relative humidity in asealed container containing a desiccant.
 209. The unit dosage formaccording to claim 208, wherein the chromatographic purity of thelinaclotide is greater than or equal to 91%, 92%, 93%, 94%, 95% or 96%after (a) 18 months of storage of the unit dosage form at 25° C. at 60%relative humidity in a sealed container containing a desiccant or (b) 6months of storage of the unit dosage form at 40° C. at 75% relativehumidity in a sealed container containing a desiccant.
 210. A sealedcontainer comprising a plurality of unit dosage forms of apharmaceutical composition comprising linaclotide and a pharmaceuticallyacceptable excipient wherein the chromatographic purity of thelinaclotide is greater than or equal to 90% after (a) 18 months ofstorage of the sealed container containing a desiccant at 25° C. at 60%relative humidity or (b) 6 months of storage of the sealed containercontaining a desiccant at 40° C. at 75% relative humidity.
 211. Thesealed container according to claim 210, wherein the chromatographicpurity of the linaclotide is greater than or equal to 91%, 92%, 93%,94%, 95% or 96% after (a) 18 months of storage of the sealed containercontaining a desiccant at 25° C. at 60% relative humidity or (b) 6months of storage of the sealed container containing a desiccant at 40°C. at 75% relative humidity.
 212. A pharmaceutical compositioncomprising linaclotide and a pharmaceutically acceptable excipient,wherein the assay value for linaclotide determined on a weight/weightbasis is greater than or equal to 90% after (a) 18 months of storage ofthe pharmaceutical composition at 25° C. at 60% relative humidity in asealed container containing a desiccant or (b) 6 months of storage ofthe pharmaceutical composition at 40° C. at 75% relative humidity in asealed container containing a desiccant.
 213. The pharmaceuticalcomposition according to claim 212, wherein the assay value for thelinaclotide is greater than or equal to 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% after (a) 18 months of storage of the pharmaceuticalcomposition at 25° C. at 60% relative humidity in a sealed containercontaining a desiccant or (b) 6 months of storage of the pharmaceuticalcomposition at 40° C. at 75% relative humidity in a sealed containercontaining a desiccant.
 214. A unit dosage form of a pharmaceuticalcomposition comprising linaclotide and a pharmaceutically acceptableexcipient, wherein the assay value for linaclotide determined on aweight/weight basis is greater than or equal to 90% after (a) 18 monthsof storage of the unit dosage form at 25° C. at 60% relative humidity ina sealed container containing a desiccant or (b) 6 months of storage ofthe unit dosage form at 40° C. at 75% relative humidity in a sealedcontainer containing a desiccant.
 215. The unit dosage form according toclaim 214, wherein the assay value for the linaclotide is greater thanor equal to 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% after (a) 18months of storage of the unit dosage form at 25° C. at 60% relativehumidity in a sealed container containing a desiccant or (b) 6 months ofstorage of the unit dosage form at 40° C. at 75% relative humidity in asealed container containing a desiccant.
 216. A sealed containercomprising a plurality of unit dosage forms of a pharmaceuticalcomposition comprising linaclotide and a pharmaceutically acceptableexcipient wherein the assay value for linaclotide in the unit dosageforms determined on a weight/weight basis is greater than or equal to90% after (a) 18 months of storage of the sealed container containing adesiccant at 25° C. at 60% relative humidity or (b) 6 months of storagethe sealed container containing a desiccant at 40° C. at 75% relativehumidity.
 217. The sealed container according to claim 216, wherein theassay value for the linaclotide is greater than or equal to 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% after (a) 18 months of storage thesealed container containing a desiccant at 25° C. at 60% relativehumidity or (b) 6 months of storage the sealed container containing adesiccant at 40° C. at 75% relative humidity.
 218. The unit dosage formaccording to any one of claim 208-209 or 214-215 or the sealed containeraccording to any one of claim 210-211 or 216-217, wherein each unitdosage form contains from 50 μg to 2 mg linaclotide.
 219. The unitdosage form or the sealed container according to claim 218, wherein eachunit dosage form contains 67.5 μg, 100 μg, 133 μg, 150 μg, 200 μg, 266μg, 300 μg, 400 μg, 500 μg or 600 μg linaclotide.
 220. Thepharmaceutical composition according to either of claim 206 or 207,wherein the chromatographic purity of the linaclotide is greater than90% after (a) 24 months of storage of the pharmaceutical composition at25° C. at 60% relative humidity in a sealed container containing adesiccant or (b) 6 months of storage of the pharmaceutical compositionat 40° C. at 75% relative humidity in a sealed container containing adesiccant.
 221. The unit dosage form according to either of claim 208 or209, wherein the chromatographic purity of the linaclotide is greaterthan 90% after (a) 24 months of storage of the unit dosage form at 25°C. at 60% relative humidity in a sealed container containing a desiccantor (b) 6 months of storage of the unit dosage form at 40° C. at 75%relative humidity in a sealed container containing a desiccant.
 222. Thesealed container according to either of claim 210 or 211, wherein thechromatographic purity of the linaclotide is greater than 90% after (a)a first 24 months of storage of the sealed container containing adesiccant at 25° C. at 60% relative humidity or (b) a first 6 months ofstorage of the sealed container containing a desiccant at 40° C. at 75%relative humidity.
 223. The pharmaceutical composition according toeither of claim 212 or 213, wherein the assay value of the linaclotideis greater than 90% after (a) a first 24 months of storage of thepharmaceutical composition at 25° C. at 60% relative humidity in asealed container containing a desiccant or (b) a first 6 months ofstorage of the pharmaceutical composition at 40° C. at 75% relativehumidity in a sealed container containing a desiccant.
 224. The unitdosage form according to either of claim 214 or 215, wherein the assayvalue of the linaclotide is greater than 90% after (a) a first 24 monthsof storage of the unit dosage form at 25° C. at 60% relative humidity ina sealed container containing a desiccant or (b) a first 6 months ofstorage of the unit dosage form at 40° C. at 75% relative humidity in asealed container containing a desiccant.
 225. The sealed containeraccording to either of claim 216 or 217, wherein the assay value of thelinaclotide is greater than 90% after (a) a first 24 months of storageof the sealed container containing a desiccant at 25° C. at 60% relativehumidity or (b) a first 6 months of storage of the sealed containercontaining a desiccant at 40° C. at 75% relative humidity.
 226. Thepharmaceutical composition according to any one of claim 206, 207 or223, the unit dosage form according to either of claim 208, 209 or 224,or the sealed container according to any one of claim 210, 211 or 225,wherein a hydrolysis product having a structure of

comprises less than 2% by weight compared to the weight of thelinaclotide.
 227. The pharmaceutical composition, unit dosage form orsealed container according to claim 226, wherein the hydrolysis productcomprises less than 1% by weight or less than 0.5% by weight compared tothe weight of the linaclotide.
 228. The pharmaceutical composition, unitdosage form or sealed container according to claim 227, wherein thehydrolysis product comprises less than 0.1% by weight or less than 0.05%by weight compared to the weight of the linaclotide.
 229. Thepharmaceutical composition according to any one of claim 206, 207 or223, the unit dosage form according to any one of claim 208, 209 or 224,or the sealed container according to any one of claim 210, 211 or 225,wherein a formaldehyde imine product having a structure of

comprises less than 2% by weight compared to the weight of thelinaclotide.
 230. The pharmaceutical composition, unit dosage form orsealed container according to claim 229, wherein the formaldehyde imineproduct comprises less than 1% by weight or less than 0.5% by weightcompared to the weight of the linaclotide.
 231. The pharmaceuticalcomposition, unit dosage form or sealed container according to claim230, wherein the formaldehyde imine product comprises less than 0.1% byweight or less than 0.05% by weight compared to the weight of thelinaclotide.
 232. The pharmaceutical composition according to any one ofclaim 206, 207 or 223, the unit dosage form according to any one ofclaim 208, 209 or 224, or the sealed container according to any one ofclaim 210, 211 or 225, wherein a linaclotide oxidation product having amolecular weight of 1542.8 comprises less than 2% by weight compared tothe weight of the linaclotide.
 233. The pharmaceutical composition, unitdosage form or sealed container according to claim 225, wherein thelinaclotide oxidation product comprises less than 1% by weight or lessthan 0.5% by weight compared to the weight of the linaclotide.
 234. Thepharmaceutical composition, unit dosage form or sealed containeraccording to claim 233, wherein the linaclotide oxidation productcomprises less than 0.1% by weight or less than 0.05% by weight comparedto the weight of the linaclotide.
 235. A unit dosage form comprising thepharmaceutical composition according to any one of claims 21-95. 236.The unit dosage form according to claim 235, wherein said unit dosageform contains 50 μg to 1 mg linaclotide.
 237. The unit dosage formaccording to claim 236, wherein said unit dosage form is a capsule ortablet and wherein each unit dosage form contains 67.5 μg, 100 μg, 133μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg or 600 μglinaclotide.
 238. A sealed container comprising a plurality of unitdosage forms according to any one of claim 96-98 or 235-237.
 239. Amethod for treating irritable bowel syndrome with constipation (IBS-c)in an adult patient in need thereof, comprising administering to thepatient once daily an effective amount of the pharmaceutical compositionaccording to any one of claims 1-95.
 240. The method according to claim239, wherein said effective amount is 266 μg linaclotide.
 241. Themethod according to claim 239, wherein said effective amount is 133 μglinaclotide.
 242. The method according to any one of claims 239-241,wherein said treating is for a period of at least one week.
 243. Themethod according to claim 242, wherein said treating is for a period ofat least four weeks.
 244. The method according to claim 239, whereinsaid treating improves at least one symptom selected from reducedabdominal pain, an increase in the number of complete spontaneous bowelmovements (CSBM) in a week, an increase in the number of spontaneousbowel movements (SBM) in a week, improved stool consistency, reducedstraining, reduced abdominal discomfort, reduced bloating or reducedIBS-c symptom severity.
 245. The method according to claim 244, whereinsaid treating improves at least one symptom selected from reducedabdominal pain, reduced abdominal discomfort or reduced bloating. 246.The method according to claim 244, wherein said treating improves atleast one symptom selected from an increase in the number of CSBM in aweek, an increase in the number of spontaneous bowel movements SBM in aweek, improved stool consistency or reduced straining.
 247. The methodaccording to any one of claims 239-246, wherein a capsule or tabletcomprises said pharmaceutical composition.
 248. A method for treatingchronic constipation in an adult human patient in need thereof,comprising administering to the patient once daily an effective amountof the pharmaceutical composition according to any one of claims 1-95.249. The method according to claim 248, wherein said effective amount is266 μg linaclotide.
 250. The method according to claim 248, wherein saideffective amount is 133 μg linaclotide.
 251. The method according to anyone of claims 248-250, wherein said treating is for a period of at leastone week.
 252. The method according to claim 251, wherein said treatingis for a period of at least four weeks.
 253. The method according toclaim 248, wherein said treating improves at least one symptom selectedfrom an increase in the number of complete spontaneous bowel movements(CSBM) in a week, an increase in the number of spontaneous bowelmovements (SBM) in a week, improved stool consistency, reducedstraining, reduced abdominal discomfort, reduced bloating or reducedseverity of constipation.
 254. The method according to claim 253,wherein said treating improves at least one symptom selected fromreduced abdominal discomfort, reduced bloating or reduced severity ofconstipation.
 255. The method according to claim 253, wherein saidtreating improves at least one symptom selected from an increase in thenumber of CSBM in a week, an increase in the number of spontaneous bowelmovements SBM in a week, improved stool consistency or reducedstraining.
 256. The method according to any one of claims 248-255,wherein a capsule or tablet comprises said pharmaceutical composition.257. A method for preparing a pharmaceutical composition comprisinglinaclotide or a salt thereof, the method comprising: (a) providing asolution comprising: (i) linaclotide or a pharmaceutically acceptablesalt thereof (ii) one or more of a cation selected from Mg²⁺, Ca²⁺,Zn²⁺, Mn²⁺, K⁺, Na⁺ or Al³⁺ or a sterically hindered primary amine; and,(b) spray drying the solution to produce linaclotide microparticles.258. The method according to claim 257, wherein said solution isaqueous.
 259. The method according to claim 257, wherein said solutionfurther comprises a binder or filler.
 260. A pharmaceutical compositioncomprising: linaclotide; Ca²⁺; leucine; and hydroxypropylmethylcellulose, wherein the linaclotide is present in thepharmaceutical composition in an amount between 100 μg to 600 μg and themolar ratio of Ca²⁺:leucine:linaclotide is between 5-100:5-50:1. 261.The pharmaceutical composition of claim 260, wherein the linaclotide ispresented in an amount of 266 μg.
 262. The pharmaceutical composition ofclaims 260-261, wherein the Ca²⁺ is provided as CaCl₂.
 263. Thepharmaceutical composition of claims 262, wherein the CaCl₂ is presentin an amount of 1541 μg.
 264. The pharmaceutical composition of claims260-263, wherein the leucine is present in an amount of 687 μg.
 265. Thepharmaceutical composition of claims 260-264, wherein the hydroxypropylmethylcellulose is present in an amount of 700 μg.
 266. A pharmaceuticalcomposition comprising coated beads, wherein the beads are coated with acoating solution comprising linaclotide.
 267. The pharmaceuticalcomposition of claim 266, wherein the coating solution comprises:linaclotide; Ca²⁺; leucine; and hydroxypropyl methylcellulose, whereinthe linaclotide is present in the pharmaceutical composition in anamount between 100 μg to 600 μg and the molar ratio the ofCa²⁺:leucine:linaclotide is between 5-100:5-50:1.
 268. Thepharmaceutical composition of claim 267, wherein the linaclotide ispresent in an amount of 266 μg.
 269. The pharmaceutical composition ofclaims 267-268, wherein the Ca²⁺ is provided as CaCl₂.
 270. Thepharmaceutical composition of claim 269, wherein the CaCl₂ is present inan amount of 1541 μg.
 271. The pharmaceutical composition of claims267-270, wherein the leucine is present in an amount of 687 μg.
 272. Thepharmaceutical composition of claims 267-271, wherein the hydroxypropylmethylcellulose is present in an amount of 700 μg.
 273. Thepharmaceutical composition of claims 267-272, wherein the beads comprisemicrocrystalline cellulose.